Lipocalin muteins with binding affinity for lag-3

ABSTRACT

The present disclosure provides human tear lipocalin muteins that specifically bind to LAG-3, which can be used in pharmaceutical applications, for example, as anti-cancer agents and/or immune modulators for the treatment or prevention of human diseases such as cancer, infectious diseases, and autoimmune diseases. The present disclosure further shows the human lipocalin muteins can inhibit the binding of LAG-3 to MHC class II on cells overexpressing MHC class II. The present disclosure also concerns methods of making LAG-3 binding lipocalin muteins described herein as well as compositions comprising such lipocalin muteins. The present disclosure further relates to nucleic acid molecules encoding such lipocalin muteins and to methods for generation of such lipocalin muteins and nucleic acid molecules. In addition, the application discloses therapeutic and/or diagnostic uses of these lipocalin muteins as well as compositions comprising one or more of such lipocalin muteins.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.16/478,465, filed Jul. 16, 2019 as a National Stage application ofPCT/EP2018/051139, filed Jan. 18, 2018, which claims priority fromEuropean Application No. 17151945,7, filed Jan. 18, 2017, each of whichare hereby incorporated by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing, which has beensubmitted in ASCII format via EFS-WEB and is hereby incorporated byreference in its entirety. Said ASCII copy, created on October 7, 2021,is named “Sequence Listing.txt” and is 120 KB in size.

I. BACKGROUND

Lymphocyte Activation Gene-3, or LAG-3 (also known as Cluster ofDifferentiation 223 or CD223) is a membrane protein of theimmunoglobulin supergene family. LAG-3 is structurally and geneticallyrelated to CD4, with its encoding gene located on the distal part of theshort arm of chromosome 12, near the CD4 gene, suggesting that the LAG-3gene may have evolved through gene duplication (Triebel et al., J ExpMed, 1990). LAG-3 is not expressed on resting peripheral bloodlymphocytes but is expressed on activated T cells and natural killer(NK) cells (Triebel et al., J Exp Med, 1990), and has been reported toalso be expressed on activated B cells (Kisielow et al., Eur J Immunol,2005) and plasmacytoid dendritic cells (Workman et al., J Immunol,2009).

Like CD4, LAG-3 binds to major histocompatibility complex (MHC) class Hmolecules, but with a higher affinity and at a different binding site(Huard et al., Proc Natl Acad Sci U S A, 1997), MHC class H engagementon dendritic cells by LAG-3 leads to changes in the cytokine andchemokine profiles of dendritic cells (Buisson and Triebel, Vaccine,2003). Further, LAG-3 has been reported to cause maturation of dendriticcells, as demonstrated by the production of 1L-12 and TNF-alpha by thesecells and increases in the capacity of dendritic cells to stimulate theproliferation and IFN-gamma response by allogeneic T-cells (Andreae etal., J Immunol, 2002). LAG-3 signaling and MHC class H cross-linking hasbeen reported to inhibit early events in primary activation of humanCD4⁺ and CD8⁺ T-cells (Macon-Lemaitre and Triebel, Immunology, 2005). Itnegatively regulates the cellular proliferation, activation, andhomeostasis of T cells.

Like CTLA-4 and PD-1, LAG-3 is an inhibitory immune receptor. LAG-3'sprominent role as a negative regulator of T cell responses has beenimpressively demonstrated in particular in conjunction with PD-1 in astudy based on both knockout mice and target-specific antibodies (Woo etal., Cancer Res, 2012). In this study, dual anti-LAG-3/anti-PD-1antibody treatment cured most mice of established tumors that werelargely resistant to single antibody treatment. Further, LAG-3/PD-1double knock-out mice showed markedly increased survival from andclearance of multiple transplantable tumors. Further strong experimentalsupport for the powerful combined role of PD-1 and LAG-3 as immunecheckpoints was provided by the fact that the double knock-out mice werehighly prone to lethal autoinflammation.

Consequently, there exists an unmet need in the art for compounds thatmodulate responses of LAG-3⁺ lymphocytes, such as T-cells, NK cells, Bcells, and plasmacytoid dendritic cells, which may have important usesin the treatment or prevention of cancer, organ transplant rejection, ortreatment of autoimmune or autoinflammatory diseases. It is furtherdesirable to have lipocalin muteins that are capable of binding LAG-3with high affinity, that have enhanced biostability, and that can beused in pharmaceutical and/or diagnostic applications. In this regard,it is an object of the present disclosure to provide such lipocalinmuteins. No such lipocalin muteins having these high binding affinitiesand enhanced biostability features have been previously described.

In addition, it has been regarded as natural that monkey metabolism isthe most similar to that of humans, and, accordingly, cynomolgus monkeyshave been widely used in pharmacokinetic or drug-safety studies in thedevelopment of new therapies, including new biologics. Such studies mayfurther be necessary prerequisites to regulatory approval. Thus, it isalso desirable to have lipocalin muteins that are cross-reactive withboth human and cynomolgus LAG-3, with comparable binding pattern,including comparable or similar binding affinity. No such lipocalinmuteins having these cross-reactivity features have been previouslydescribed.

The recitation of any reference in this application is not an admissionthat the reference is prior art to this application.

In this regard, the present disclosure provides a group of novelcompounds specifically binding to the LAG-3 of both humans andcynomolgus monkeys with high affinity and with enhanced biostabilityfeatures, thereby, modulating the immune response. Such compounds aremuteins derived from lipocalins and may be used in pharmaceutical,diagnostic or other applications. Muteins of lipocalins are a rapidlyexpanding class of therapeutics and can be constructed to exhibit highaffinity and specificity against desired targets (see, e.g.,International Patent Publication Nos. WO 99/16873, WO 00/75398, WO03/029471, and WO 05/19256).

II. DEFINITIONS

The following list defines terms, phrases, and abbreviations usedthroughout the instant specification. All terms listed and definedherein are intended to encompass all grammatical forms.

As used herein, unless otherwise specified, “LAG-3” means human LAG-3(huLAG-3) and includes variants, isoforms and species homologs of humanLAG-3. LAG-3 is also known as “lymphocyte-activation gene 3”, “duster ofdifferentiation 223” or “CD223”, which are used interchangeably. HumanLAG-3 means a full-length protein defined by UniProt P18627 (version 5of 7 Jul. 2009), a fragment thereof, or a variant thereof. CynomolgusLAG-3 (cyLAG-3) refers to the LAG-3 of cynomolgus monkeys. CyLAG-3 mayalso be used to refer to the extracellular domain of cyLAG-3 as setforth in position 1-428 of SEQ ID NO: 56.

As used herein, “detectable affinity” means the ability to bind to aselected target with an affinity, generally measured by K_(d) or EC₅₀,of at most about 10⁻⁵ M or below (a lower K_(d) or EC₅₀ value reflectsbetter binding activity). Lower affinities are generally no longermeasurable with common methods such as ELISA (enzyme-linkedimmunosorbent assay) and therefore of secondary importance.

As used herein, “binding affinity” of a protein of the disclosure (e.g.,a mutein of a lipocalin) or a fusion polypeptide thereof to a selectedtarget (in the present case, LAG-3), can be measured (and thereby K_(d)values of a mutein-ligand complex can be determined) by a multitude ofmethods known to those skilled in the art. Such methods include, but arenot limited to, fluorescence titration, competitive ELISA, calorimetricmethods, such as isothermal titration calorimetry (ITC), and surfaceplasmon resonance (SPR). Such methods are well established in the artand examples thereof are also detailed below.

It is also noted that the complex formation between the respectivebinder and its ligand is influenced by many different factors such asthe concentrations of the respective binding partners, the presence ofcompetitors, pH and the ionic strength of the buffer system used, andthe experimental method used for determination of the dissociationconstant K_(d) (for example fluorescence titration, competition ELISA orsurface plasmon resonance, just to name a few) or even the mathematicalalgorithm which is used for evaluation of the experimental data.

Therefore, it is also clear to the skilled person that the K_(d) values(dissociation constant of the complex formed between the respectivebinder and its target/ligand) may vary within a certain experimentalrange, depending on the method and experimental setup that is used fordetermining the affinity of a particular lipocalin mutein for a givenligand. This means that there may be a slight deviation in the measuredK_(d) values or a tolerance range depending, for example, on whether theK_(d) value was determined by surface plasmon resonance (SPR), bycompetitive ELISA, or by direct ELISA.

As used herein, a “mutein,” a “mutated” entity (whether protein ornucleic acid), or “mutant” refers to the exchange, deletion, orinsertion of one or more nucleotides or amino acids, compared to thenaturally occurring (wild-type) nucleic acid or protein “reference”scaffold. Said term also includes fragments of a mutein and variants asdescribed herein. Lipocalin muteins of the present invention, fragmentsor variants thereof preferably have the function of binding to LAG-3 asdescribed herein.

The term “fragment” as used herein in connection with the muteins of thedisclosure relates to proteins or peptides derived from full-lengthmature human tear lipocalin (hTlc or hTLPC) that is N-terminally and/orC-terminally shortened, i.e., lacking at least one of the N-terminaland/or C-terminal amino acids. Such a fragment may lack up to 2, up to3, up to 4, up to 5, up to 10, up to 15, up to 20, up to 25, or up to 30(including all numbers in between) of the N-terminal and/or C-terminalamino acids. As an illustrative example, such a fragment may lack 4N-terminal and 2 C-terminal amino acids. It is understood that thefragment is preferably a functional fragment of the full-length tearlipocalin (mutein), which means that it preferably comprises the bindingpocket of the full-length tear lipocalin (mutein) it is derived from. Asan illustrative example, such a functional fragment may comprise atleast amino acids 5-156 of the linear polypeptide sequence of nativemature human tear lipocalin. Such fragments may include at least 10,more such as 20 or 30 or more consecutive amino acids of the primarysequence of mature tear lipocalin and are usually detectable in animmunoassay of the mature lipocalin.

In general, the term “fragment,” as used herein with respect to thecorresponding protein ligand LAG-3 of a lipocalin mutein of thedisclosure or of the combination according to the disclosure or of afusion protein described herein, relates to N-terminally and/orC-terminally shortened protein or peptide ligands, which retain thecapability of the full-length ligand to be recognized and/or bound by amutein according to the disclosure.

The term “mutagenesis” as used herein means that the experimentalconditions are chosen such that the amino acid naturally occurring at agiven sequence position of the mature lipocalin can be substituted by atleast one amino acid that is not present at this specific position inthe respective natural polypeptide sequence. The term “mutagenesis” alsoincludes the (additional) modification of the length of sequencesegments by deletion or insertion of one or more amino acids. Thus, itis within the scope of the disclosure that, for example, one amino acidat a chosen sequence position is replaced by a stretch of three randommutations, leading to an insertion of two amino acid residues comparedto the length of the respective segment of the wild-type protein. Suchan insertion or deletion may be introduced independently from each otherin any of the peptide segments that can be subjected to mutagenesis inthe disclosure. In one exemplary embodiment of the disclosure, aninsertion of several mutations may be introduced into the loop AB of thechosen lipocalin scaffold (cf. International Patent Publication No. WO2005/019256 which is incorporated by reference its entirety herein).

The term “random mutagenesis” means that no predetermined single aminoacid (mutation) is present at a certain sequence position but that atleast two amino acids can be incorporated with a certain probability ata predefined sequence position during mutagenesis.

“Identity” is a property of sequences that measures their similarity orrelationship. The term “sequence identity” or “identity” as used in thepresent disclosure means the percentage of pair-wise identicalresidues—following (homologous) alignment of a sequence of a polypeptideof the disclosure with a sequence in question—with respect to the numberof residues in the longer of these two sequences. Sequence identity ismeasured by dividing the number of identical amino acid residues by thetotal number of residues and multiplying the product by 100.

The term “homology” is used herein in its usual meaning and includesidentical amino acids as well as amino acids which are regarded to beconservative substitutions (for example, exchange of a glutamate residueby an aspartate residue) at equivalent positions in the linear aminoacid sequence of a polypeptide of the disclosure (e.g., any lipocalinmuteins of the disclosure).

The percentage of sequence homology or sequence identity can, forexample, be determined herein using the program BLASTP, version blastp2.2.5 (Nov. 16, 2002) (cf. Altschul et al., Nucleic Acids Res, 1997). Inthis embodiment, the percentage of homology is based on the alignment ofthe entire polypeptide sequence (matrix: BLOSUM 62; gap costs: 11.1;cut-off value set to 10⁻³) including the propeptide sequences,preferably using the wild-type protein scaffold as reference in apairwise comparison. It is calculated as the percentage of numbers of“positives” (homologous amino acids) indicated as result in the BLASTPprogram output divided by the total number of amino acids selected bythe program for the alignment.

Specifically, in order to determine whether an amino acid residue of theamino acid sequence of a lipocalin (mutein) is different from awild-type lipocalin corresponding to a certain position in the aminoacid sequence of a wild-type lipocalin, a skilled artisan can use meansand methods well-known in the art, e.g., alignments, either manually orby using computer programs such as BLAST 2.0, which stands for BasicLocal Alignment Search Tool, or ClustalW, or any other suitable programwhich is suitable to generate sequence alignments. Accordingly, awild-type sequence of lipocalin can serve as “subject sequence” or“reference sequence,” while the amino acid sequence of a lipocalindifferent from the wild-type lipocalin described herein serves as “querysequence.” The terms “wild-type sequence” and “reference sequence” and“subject sequence” are used interchangeably herein. A preferredwild-type sequence of lipocalin is the sequence of hTlc as shown in SEQID NO: 1.

“Gaps” are spaces in an alignment that are the result of additions ordeletions of amino acids. Thus, two copies of exactly the same sequencehave 100% identity, but sequences that are less highly conserved, andhave deletions, additions, or replacements, may have a lower degree ofsequence identity. Those skilled in the art will recognize that severalcomputer programs are available for determining sequence identity usingstandard parameters, for example BLAST (Altschul, et al. (1997) NucleicAcids Res. 25, 3389-3402), Blast2 (Altschul, et al. (1990) J. Mol. Biol.215, 403-410), and Smith-Waterman (Smith, et al. (1981) J. Mol. Biol.147, 195-197).

The term “variant” as used in the present disclosure relates toderivatives of a protein or peptide that include modifications of theamino acid sequence, for example by substitution, deletion, insertion orchemical modification. Such modifications do in some embodiments notreduce the functionality of the protein or peptide. Such variantsinclude proteins, wherein one or more amino acids have been replaced bytheir respective D-stereoisomers or by amino acids other than thenaturally occurring 20 amino acids, such as, for example, ornithine,hydroxyproline, citrulline, homoserine, hydroxylysine, norvaline.However, such substitutions may also be conservative, i.e., an aminoacid residue is replaced with a chemically similar amino acid residue.Examples of conservative substitutions are the replacements among themembers of the following groups: 1) alanine, serine, and threonine; 2)aspartic acid and glutamic acid; 3) asparagine and glutamine; 4)arginine and lysine; 5) isoleucine, leucine, methionine, and valine; and6) phenylalanine, tyrosine, and tryptophan. The term “variant,” as usedherein with respect to the corresponding protein target LAG-3 of alipocalin mutein of the disclosure or of a combination and/or a fusionprotein according to the disclosure, relates to LAG-3 or fragmentthereof, that has one or more such as 1, 2, 3, 4 ,5 ,6, 7, 8, 9, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 40, 50, 60, 70, 80 or more aminoacid substitutions, deletions and/or insertions in comparison to awild-type LAG-3 protein, such as a LAG-3 reference protein as depositedwith UniProt as described herein. A LAG-3 variant has preferably anamino acid identity of at least 50%, 60%, 70%, 80%, 85%, 90% or 95% witha wild-type LAG-3, such as a human LAG-3 reference protein as depositedwith UniProt as described herein.

By a “native sequence” of a lipocalin is meant that the sequence of alipocalin that has the same amino acid sequence as the correspondingpolypeptide derived from nature. Thus, a native sequence lipocalin canhave the amino acid sequence of the respective naturally-occurringlipocalin from any organism, in particular, a mammal. Such nativesequence polypeptide can be isolated from nature or can be produced byrecombinant or synthetic means. The term “native sequence” polypeptidespecifically encompasses naturally-occurring truncated or secreted formsof the lipocalin, naturally-occurring variant forms such asalternatively spliced forms and naturally-occurring allelic variants ofthe lipocalin. A polypeptide “variant” means a biologically activepolypeptide having at least about 50%, 60%, 70%, 80% or at least about85% amino acid sequence identity with the native sequence polypeptide.Such variants include, for instance, polypeptides in which one or moreamino acid residues are added or deleted at the N- or C-terminus of thepolypeptide. Generally, a variant has at least about 70%, including atleast about 80%, such as at least about 85% amino acid sequenceidentity, including at least about 90% amino acid sequence identity orat least about 95% amino acid sequence identity with the native sequencepolypeptide. As an illustrative example, the first 4 N-terminal aminoacid residues (His-His-Leu-Leu, SEQ ID NO: 51) and the last 2 C-terminalamino acid residues (Ser-Asp) can be deleted or mutated in a hTlc muteinof the disclosure without affecting the biological function of theprotein, e.g., SEQ ID NOs: 7-28, 57-70, and 85-95.

The term “position” when used in accordance with the disclosure meansthe position of either an amino acid within an amino acid sequencedepicted herein or the position of a nucleotide within a nucleic acidsequence depicted herein. To understand the term “correspond” or“corresponding” as used herein in the context of the amino acid sequencepositions of one or more lipocalin muteins, a corresponding position isnot only determined by the number of the preceding nucleotides/aminoacids. Accordingly, the position of a given amino acid in accordancewith the disclosure which may be substituted may vary due to deletion oraddition of amino acids elsewhere in a (mutant or wild-type) lipocalin.Similarly, the position of a given nucleotide in accordance with thepresent disclosure which may be substituted may vary due to deletions oradditional nucleotides elsewhere in a mutein or wild-type lipocalin5′-untranslated region (UTR) including the promoter and/or any otherregulatory sequences or gene (including exons and introns).

Thus, for a “corresponding position” in accordance with the disclosure,it is preferably to be understood that the positions ofnucleotides/amino acids may differ in the indicated number than similarneighboring nucleotides/amino acids, but said neighboringnucleotides/amino acids, which may be exchanged, deleted, or added, arealso comprised by the one or more “corresponding positions”.

In addition, for a corresponding position in a lipocalin mutein based ona reference sequence in accordance with the disclosure, it is preferablyunderstood that the positions of nucleotides/amino acids structurallycorrespond to the positions elsewhere in a (mutant or wild-type)lipocalin, even if they may differ in the indicated number, asappreciated by the skilled in light of the highly-conserved overallfolding pattern among lipocalins.

The term “albumin” includes all mammal albumins such as human serumalbumin or bovine serum albumin or rat serum albumin.

The term “organic molecule” or “small organic molecule” as used hereinfor the non-natural target denotes an organic molecule comprising atleast two carbon atoms, but preferably not more than 7 or 12 rotatablecarbon bonds, having a molecular weight in the range between 100 and2,000 Daltons, preferably between 100 and 1,000 Daltons, and optionallyincluding one or two metal atoms.

The word “detect,” “detection,” “detectable,” or “detecting” as usedherein is understood both on a quantitative and a qualitative level, aswell as a combination thereof. It thus includes quantitative,semi-quantitative and qualitative measurements of a molecule ofinterest.

A “subject” is a vertebrate, preferably a mammal, more preferably ahuman. The term “mammal” is used herein to refer to any animalclassified as a mammal, including, without limitation, humans, domesticand farm animals, and zoo, sports, or pet animals, such as sheep, dogs,horses, cats, cows, rats, pigs, apes such as cynomolgus monkeys, to nameonly a few illustrative examples. Preferably, the “mammal” herein ishuman.

An “effective amount” is an amount sufficient to effect beneficial ordesired results. An effective amount can be administered in one or moreadministrations.

A “sample” is defined as a biological sample taken from any subject.Biological samples include, but are not limited to, blood, serum, urine,feces, semen, or tissue.

III. DESCRIPTIONS OF FIGURES

FIG. 1: depicts an alignment of amino acid sequences of optimized LAG-3specific human tear lipocalin (hTlc) muteins, in comparison with thelinear polypeptide sequence of mature hTlc. Compared to the linearpolypeptide sequence of mature hTlc (SEQ ID NO: 1), the first 4N-terminal amino acid residues (His, His, Leu, Leu; SEQ ID NO: 50) andthe last 2 C-terminal amino acid residues (Ser, Asp) are deleted inthese hTlc-derived, LAG-3-binding muteins (listed as hTlc muteins SEQ IDNOs: 7-28 and 57-70) and the negative-control muteins (SEQ ID NOs: 3 and4).

FIG. 2A and FIG. 2B: depict the results of fluorescence-activated cellsorting (FACS) studies carried out in order to assess the specificbinding of the lipocalin muteins to human LAG-3 (FIG. 2A) and cynomolgusLAG-3 (FIG. 2B), respectively, expressed on mammalian cells as describedin Example 5. Chinese hamster ovary (CHO) cells stably transfected withhuman or cynomolgus LAG-3 were incubated with lipocalin muteins, and thebound muteins were detected using a fluorescently labeled anti-hTlcantibody. All lipocalin muteins show binding to LAG-3 expressed on CHOcells with EC₅₀ comparable to tested benchmark antibody (SEQ ID NOs: 5and 6). The negative lipocalin mutein SEQ ID NO: 3 and negative controlhuman IgG4 (hIgG4) (SEQ ID NOs: 55 and 56, Sigma #I4639) showed nobinding. The geometric means of the fluorescence intensity werenormalized to the maximal mean and fit with a 1:1 binding model. Theresulting EC₅₀ values are provided in Table 3.

FIG. 3: shows that lipocalin muteins compete with majorhistocompatibility complex (MHC) class II molecules (LAG-3′s naturalligands) for the binding to LAG-3 in a competitive FACS experiment. MHCclass II positive human cell line A375 was incubated with lipocalinmutein and huLAG-3-Fc (human LAG-3 extracellular domain fused to humanIgG1 Fc fragment, R&D systems), the bound huLAG-3-Fc was detected usinga goat anti-human IgG antibody conjugated with phycoerythrin (JacksonImmunoResearch Laboratories Inc., #109-1 16-098). A dose dependentinhibition of huLAG-3-Fc binding to MHC class II molecules by LAG-3specific lipocalin muteins was shown. The LAG-3 specific lipocalinmuteins and the reference molecule (SEQ ID NOs: 5 and 6) showedinhibitory effect on LAG-3/MHC class II binding at equal concentrations.The negative control lipocalin mutein (SEQ ID NO: 3) and hIgG4 negativecontrol did not lead to measurable inhibition of huLAG-3-Fc binding toA375 cells expressing MHC class II molecules.

IV. DETAILED DESCRIPTION OF THE DISCLOSURE

As used herein, a “lipocalin” is defined as a monomeric protein ofapproximately 18-20 kDa in weight, having a cylindrical p-pleated sheetsupersecondary structural region comprising a plurality of (preferablyeight) p-strands connected pair-wise by a plurality of (preferably four)loops at one end to define thereby a binding pocket. It is the diversityof the loops in the otherwise rigid lipocalin scaffold that gives riseto a variety of different binding modes among the lipocalin familymembers, each capable of accommodating targets of different size, shape,and chemical character (reviewed, e.g., in Skerra, Biochim Biophys Acta,2000, Flower et al., Biochim Biophys Acta, 2000, Flower, Biochem J,1996). Indeed, the lipocalin family of proteins have naturally evolvedto bind a wide spectrum of ligands, sharing unusually low levels ofoverall sequence conservation (often with sequence identities of lessthan 20%) yet retaining a highly conserved overall folding pattern. Thecorrespondence between positions in various lipocalins is well known toone of skill in the art (see, e.g., U.S. Pat. No. 7,250,297).

As noted above, a lipocalin is a polypeptide defined by itssupersecondary structure, namely cylindrical p-pleated sheetsupersecondary structural region comprising eight β-strands connectedpair-wise by four loops at one end to define thereby a binding pocket.The present disclosure is not limited to lipocalin muteins specificallydisclosed herein. In this regard, the disclosure relates to lipocalinmuteins having a cylindrical β-pleated sheet supersecondary structuralregion comprising eight β-strands connected pair-wise by four loops atone end to define thereby a binding pocket, wherein at least one aminoacid of each of at least three of said four loops has been mutated ascompared to the reference sequence, and wherein said lipocalin iseffective to bind LAG-3 with detectable affinity.

In one particular embodiment, a lipocalin mutein disclosed herein is amutein of human tear lipocalin (hTlc or hTLPC), also termed lipocalin-1,human tear prealbumin or von Ebner gland protein. The term “human tearlipocalin” or “hTlc” or “lipocalin-1” as used herein refers to themature human tear lipocalin with the SWISS-PROT/UniProt Data BankAccession Number P31025 (Isoform 1). The amino acid sequence shown inSWISS-PROT/UniProt Data Bank Accession Number P31025 may be used as apreferred “reference sequence,” more preferably the amino acid sequenceshown in SEQ ID NO: 1 is used herein as “reference sequence.”

In some embodiments, a lipocalin mutein binding LAG-3 with detectableaffinity may include at least one amino acid substitution of a nativecysteine residue of the reference sequence by another amino acid, forexample, a serine residue. In some other embodiments, a lipocalin muteinbinding LAG-3 with detectable affinity may include one or morenon-native cysteine residues substituting one or more amino acids of awild-type lipocalin. In a further particular embodiment, a lipocalinmutein according to the disclosure includes at least two amino acidsubstitutions of a native amino acid by a cysteine residue, hereby toform one or more cysteine bridges. In some embodiments, said cysteinebridge may connect at least two loop regions. The definition of theseregions is used herein in accordance with Flower (Biochem J, 1996),(Biochim Biophys Acta, 2000) and Breustedt et al. (J Biol Chem, 2005).In a related embodiment, the disclosure teaches one or more lipocalinmuteins that are capable of activating downstream signaling pathways ofLAG-3 by binding to LAG-3.

Proteins of the disclosure, which are directed against or specific forLAG-3, include any number of specific-binding protein muteins that arebased on a defined protein scaffold, preferably a lipocalin scaffold.Also preferably, the number of nucleotides or amino acids, respectively,that is exchanged, deleted or inserted is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more such as 25, 30, 35, 40,45 or 50, with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 being preferred and9, 10 or 11 being even more preferred. However, it is preferred thatprotein muteins of the disclosure is still capable of binding LAG-3.

In one aspect, the present disclosure includes various lipocalin muteinsthat bind LAG-3 with at least detectable affinity. In this sense, LAG-3can be regarded as a non-natural ligand of wild-type lipocalins, where“non-natural ligand” refers to a compound that does not bind towild-type lipocalin under physiological conditions. By engineeringwild-type lipocalin with one or more mutations at certain sequencepositions, the present inventors have demonstrated that high affinityand high specificity for the non-natural ligand, LAG-3, is possible. Insome embodiments, at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or even morenucleotide triplet(s) encoding certain sequence positions on wild typelipocalins, random mutagenesis may be carried out through substitutionat these positions by a subset of nucleotide triplets.

Further, the lipocalin muteins of the disclosure may have a mutatedamino acid residue at any one or more, including at least at any 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more of the sequence positionscorresponding to certain sequence positions of the linear polypeptidesequence of the reference lipocalin.

A protein of the disclosure may include the wild-type (natural) aminoacid sequence of the “parental” protein scaffold (such as a lipocalinscaffold) outside the mutated amino acid sequence positions. In someembodiments, a lipocalin mutein according to the disclosure may alsocarry one or more amino acid mutations at one or more sequenceposition(s) as long as such a mutation does, at least essentially nothamper or not interfere with the binding activity and the folding of themutein. Such mutations can be accomplished very easily on DNA levelusing established standard methods (Sambrook and Russell, 2001,Molecular cloning: a laboratory manual). Illustrative examples ofalterations of the amino acid sequence are insertions or deletions aswell as amino acid substitutions. Such substitutions may beconservative, i.e., an amino acid residue is replaced with an amino acidresidue of chemically similar properties, in particular with regard topolarity as well as size. Examples of conservative substitutions are thereplacements among the members of the following groups: 1) alanine,serine, and threonine; 2) aspartic acid and glutamic acid; 3) asparagineand glutamine; 4) arginine and lysine; 5) iso-leucine, leucine,methionine, and valine; and 6) phenylalanine, tyrosine, and tryptophan.On the other hand, it is also possible to introduce non-conservativealterations in the amino acid sequence. In addition, instead ofreplacing single amino acid residues, it is also possible to eitherinsert or delete one or more continuous amino acids of the primarystructure of the reference lipocalin, preferably as hTlc, as long asthese deletions or insertion result in a stable, folded and functionalmutein. In such mutein, for instance, one or more amino acid residuesare added or deleted at the N- or C-terminus of the polypeptide (forexample, hTlc muteins with truncated N- and C-terminus). Generally, sucha mutein may have about at least 70%, including at least about 80%, suchas at least about 85% amino acid sequence identity, with the amino acidsequence of hTlc (SEQ ID NO: 1). As an illustrative example, the presentdisclosure also encompasses hTlc muteins as defined above, in which thefirst four N-terminal amino acid residues of the sequence of maturehuman tear lipocalin (His-His-Leu-Leu; SEQ ID NO: 51; positions 1-4)and/or the last two C-terminal amino acid residues (Ser-Asp; positions157-158) of the linear polypeptide sequence of the mature human tearlipocalin have been deleted (see, e.g., SEQ ID Nos: 7-28,57-70, and85-95).

The amino acid sequence of a lipocalin mutein disclosed herein has ahigh sequence identity to the reference lipocalin, preferably hTlc, whencompared to sequence identities with other lipocalins. In this generalcontext, the amino acid sequence of a lipocalin mutein of the disclosureis at least substantially similar to the amino acid sequence of thereference lipocalin, with the proviso that possibly there are gaps (asdefined below) in an alignment that are the result of additions ordeletions of amino acids. A respective sequence of a lipocalin mutein ofthe disclosure, being substantially similar to the sequences of thereference lipocalin, has, in some embodiments, at least 70% identity orsequence homology, at least 75% identity or sequence homology, at least80% identity or sequence homology, at least 82% identity or sequencehomology, at least 85% identity or sequence homology, at least 87%identity or sequence homology, or at least 90% identity or sequencehomology including at least 95% identity or sequence homology, to thesequence of the reference lipocalin, with the proviso that the alteredposition or sequence is retained and that one or more gaps are possible.

As used herein, a lipocalin mutein of the disclosure “specificallybinds” a target (for example, LAG-3) if it is able to discriminatebetween that target and one or more reference targets, since bindingspecificity is not an absolute, but a relative property. “Specificbinding” can be determined, for example, in accordance with westernblots, ELISA, FACS, RIA (radioimmunoassay), ECL(electrochemiluminescence), IRMA (immunoradiometric assay), IHC(Immunohistochemistry), and peptide scans.

In one embodiment, the lipocalin muteins of the disclosure are fused atits N-terminus and/or its C-terminus to a fusion partner, which is aprotein domain that extends the serum half-life of the mutein. Infurther particular embodiments, the protein domain is an Fc part of animmunoglobulin, a C_(H)3 domain of an immunoglobulin, a C_(H)4 domain ofan immunoglobulin, an albumin binding peptide or an albumin bindingprotein.

In another embodiment, the lipocalin muteins of the disclosure areconjugated to a compound that extends the serum half-life of the mutein.More preferably, the muteins are conjugated to a compound selected fromthe group consisting of a polyalkylene glycol molecule, a hydroxyethylstarch, an Fc part of an immunoglobulin, a C_(H)3 domain of animmunoglobulin, a C_(H)4 domain of an immunoglobulin, an albumin bindingpeptide, and an albumin binding protein.

In yet another embodiment, the current disclosure relates to nucleicacid molecules comprising nucleotide sequences encoding lipocalinmuteins disclosed herein. The disclosure encompasses a host cellcontaining said nucleic acid molecule.

A. Lipocalin Muteins Specific for LAG-3

In one aspect, the present disclosure provides human lipocalin muteinsthat bind to human LAG-3 with high affinity and useful applications ofsuch muteins. The disclosure also provides methods of making LAG-3binding proteins described herein as well as compositions comprisingsuch proteins. LAG-3 binding proteins of the disclosure, as well ascompositions thereof, may be used in methods of detecting LAG-3 proteinin a sample or in methods of binding of LAG-3 in a subject to stimulateor inhibit immune responses. The disclosed LAG-3 binding proteins haveenhanced biostability and have a similar or comparable binding patternto both human and cynomolgus LAG-3. Finally, the disclosure providesmethods of using the muteins of lipocalin against LAG-3 to inhibit thebinding of LAG-3 to major histocompatibility complex (MHC) class IImolecules. No such human lipocalin muteins having these featuresattendant to the uses provided by present disclosure have beenpreviously described.

1. Exemplary Lipocalin Muteins Specific for LAG-3

Some embodiments of the current disclosure relate to a lipocalin muteinthat is capable of binding LAG-3, preferably human LAG-3 (huLAG-3), withan affinity measured by a K_(d) of about 80 nM, 60 nM, 40 nM, 20 nM, 15nM, 10 nM, 8 nM, 6 nM, 4 nM, 2 nM, 1.5 nM, 1 nM, 0.2 nM, 0.1 nM, 0.05nM, or even lower. Such affinity can be determined, for example, bysurface plasmon resonance (SPR) analysis essentially described inExample 4.

In other embodiments, the LAG-3 binding lipocalin mutein may becross-reactive with cynomolgus LAG-3 (cyLAG-3), and in some furtherembodiments, capable of binding cyLAG-3 with an affinity measured by aK_(d) of about 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM, or even lower such as about 0.46 nM.Such affinity can be determined, for example, by SPR analysisessentially described in Example 4.

In other embodiments, the lipocalin mutein is capable of binding LAG-3on Chinese hamster ovary (CHO) cells transfected with huLAG-3 with anEC₅₀ value of about 5 nM, 4 nM, 3 nM, 2.5 nM, 2 nM, 1.5 nM, 1 nM, 0.5 nMor even lower such as about 0.22 nM or 0.02 nM. In other embodiments,the lipocalin mutein is capable of binding LAG-3 on CHO cellstransfected with cyLAG-3 with an EC₅₀ of about 350 nM, 300 nM, 250 nM,200 nM, 150 nM, 100 nM, 50 nM, 20 nM, 10 nM, or even lower such as about9.3 nM. The EC₅₀ value can, for example, be determined by afluorescence-activated cell sorting (FACS) as essentially described inExample 5.

In some embodiments, the lipocalin mutein is capable of inhibiting thebinding of LAG-3 to MHC class II, such as those expressed onantigen-presenting cells (APCs) or tumor cells. The inhibitory mode ofaction can, for example, be determined by a FACS analysis as essentiallydescribed in Example 6.

In one aspect, the present disclosure provides LAG-3-binding hTlcmuteins.

In this regard, the disclosure provides one or more hTlc muteins thatare capable of binding LAG-3 with an affinity measured by a K_(d) ofabout 10 nM or lower, 5 nM or lower, 4 nM or lower, 3 nM or lower, 2 nMor lower, 1.5 nM or lower, 1 nM or lower, 0.75 nM or lower, 0.5 nM orlower, 0.25 nM or lower, 0.1 nM or lower, or even about 0.05 nM orlower.

In some embodiments, such hTlc mutein comprises mutated amino acidresidue(s) at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more positionscorresponding to positions 5, 7-8, 10, 14, 16, 25-34, 44, 46, 52-53,55-56, 58, 60-61, 63, 65-66, 69-70, 73, 79-80, 84-86, 89-90, 93, 96-98,101, 105-106, 108, 110-114, 121, 124, 148-150, and 152-154of the linearpolypeptide sequence of mature hTlc (SEQ ID NO: 1).

In some particular embodiments, such hTlc muteins may contain mutatedamino acid residue(s) at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or morepositions corresponding to positions 5, 7-8, 10, 16, 26-34, 44, 46, 53,56, 58, 60-61, 63, 65, 69-70, 73, 79-80, 85, 89-90, 93, 96-98, 101,105-106, 108, 111, 114, 124, 148-150, and 152-154 of the linearpolypeptide sequence of mature hTlc (SEQ ID NO: 1).

In some particular embodiments, such hTlc muteins may contain mutatedamino acid residue(s) at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, or 22 positions corresponding to positions14, 25-26, 28, 31-32, 52, 55, 58, 66, 79, 84, 86, 101, 105-106, 108,110, 112-114, and 121 of the linear polypeptide sequence of mature hTlc(SEQ ID NO: 1).

In some particular embodiments, such hTlc muteins may include mutatedamino acid residue(s) at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 positionscorresponding to positions 5, 8, 26-34, 56, 58, 60-61, 65, 69, 85, 101,105-106, 108, 111, 114, and 153-154 of the linear polypeptide sequenceof mature hTlc (SEQ ID NO: 1).

In some particular embodiments, such hTlc muteins may include mutatedamino acid residue(s) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, or 20 positions corresponding to positions 14, 25-26,28, 32, 52, 55, 58, 66, 79, 84, 86, 101, 105, 106, 108, 110, 112, 114,and 121 of the linear polypeptide sequence of mature hTlc (SEQ ID NO:1).

In some particular embodiments, such hTlc muteins may include mutatedamino acid residue(s) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, or 22 positions corresponding to positions26-34, 56, 58, 60-61, 65, 70, 101, 105-106, 108, 111, 114, and 153 ofthe linear polypeptide sequence of mature hTlc (SEQ ID NO: 1).

In some particular embodiments, such hTlc muteins may include mutatedamino acid residue(s) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, or 20 positions corresponding to positions 26-34, 56,58, 60-61, 63, 65, 101, 105-106, 108, 111, 114, 149, and 153 of thelinear polypeptide sequence of mature hTlc (SEQ ID NO: 1).

In some particular embodiments, such hTlc muteins may include mutatedamino acid residue(s) at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 positionscorresponding to positions 5, 7-8, 10, 16, 44, 46, 63, 65, 69-70, 73,80, 84, 89-90, 93, 96-98, 113, 124, 148-150, 152, or 154 of the linearpolypeptide sequence of mature hTlc (SEQ ID NO: 1).

In some further embodiments, the hTlc muteins may comprise at least 1,2, 3, 4, 5, or 6 mutated amino acid residue(s) at one or more sequencepositions corresponding to sequence positions 5, 8, 65, 69, 85, and 154of the linear polypeptide sequence of mature hTlc (SEQ ID NO: 1), andwherein said polypeptide binds LAG-3, in particular huLAG-3.

In some further embodiments, the hTlc muteins may comprise at least 1,2, or 3 mutated amino acid residue(s) at one or more sequence positionscorresponding to sequence positions 63, 65, and 149 of the linearpolypeptide sequence of mature hTlc (SEQ ID NO: 1), and wherein saidpolypeptide binds LAG-3, including huLAG-3.

In some further embodiments, the hTlc muteins may comprise at least 1,2, 3, or 4 mutated amino acid residue(s) at one or more sequencepositions corresponding to sequence positions 26, 84, 106, and 112 ofthe linear polypeptide sequence of mature hTlc (SEQ ID NO: 1), andwherein said polypeptide binds LAG-3, including huLAG-3.

In some still further embodiments, the disclosure relates to apolypeptide, wherein said polypeptide is a hTlc mutein, in comparisonwith the linear polypeptide sequence of hTlc (SEQ ID NO: 1), comprisingat least 1 mutated amino acid residue(s) at the sequence position 84,and wherein said polypeptide binds LAG-3, in particular huLAG-3.

In some embodiments, a lipocalin mutein according to the disclosure mayinclude at least one amino acid substitution of a native cysteineresidue by, e.g., a serine residue. In some embodiments, a hTlc muteinaccording to the disclosure includes an amino acid substitution of anative cysteine residue at positions 61 and/or 153 by another amino acidsuch as a serine residue. In this context it is noted that it has beenfound that removal of the structural disulfide bond (on the level of arespective nave nucleic acid library) of wild-type tear lipocalin thatis formed by the cysteine residues 61 and 153 (cf. Breustedt et al., JBiol Chem, 2005) may provide hTlc muteins that are not only stablyfolded but are also able to bind a given non-natural ligand with highaffinity. In some particular embodiments, the hTlc mutein according tothe disclosure includes the amino acid substitutions Cys 61→Ala, Phe,Lys, Arg, Thr, Asn, Gly, Gln, Asp, Asn, Leu, Tyr, Met, Ser, Pro or Trp,and/or Cys 153→Ser or Ala. Such substitutions have proven useful toprevent the formation of the naturally occurring disulphide bridgelinking Cys 61 and Cys 153, and thus to facilitate handling of themutein. However, hTlc muteins that bind LAG-3 and that have thedisulphide bridge formed between Cys 61 and Cys 153 are also part of thepresent disclosure.

In some embodiments, the elimination of the structural disulfide bondmay provide further advantage of allowing for the (spontaneous)generation or deliberate introduction of non-natural artificialdisulfide bonds into muteins of the disclosure, thereby increasing thestability of the muteins. For example, in some embodiments, either twoor all three of the cysteine codons at position 61, 101 and 153 arereplaced by a codon of another amino acid. Further, in some embodiments,a hTlc mutein according to the disclosure includes an amino acidsubstitution of a native cysteine residue at position 101 by a serineresidue or a histidine residue.

However, hTlc muteins that bind LAG-3 and that have the disulfide bridgeformed between Cys 61 and Cys 153 are also part of the presentdisclosure. In some particular embodiments, hTlc muteins that do notinclude mutated amino acids at positions 61 and 153 and have thedisulfide bond formed between Cys 61 and Cys 153. In some furtherparticular embodiments, the hTlc muteins with mutated amino acid(s) atposition(s) 61 and/or 153 are subjected to further mutagenesis torestore the natural disulfide bond by back mutating positions 61 and/or153 to the native cysteine.

In some embodiments, a mutein according to the disclosure includes anamino acid substitution of a native amino acid by a cysteine residue atpositions 28 or 105 with respect to the amino acid sequence of hTlc (SEQID NO: 1).

Further, in some embodiments, a mutein according to the disclosureincludes an amino acid substitution of a native arginine residue atpositions 111 by a proline residue with respect to the amino acidsequence of hTlc (SEQ ID NO: 1). Further, in some embodiments, a muteinaccording to the disclosure includes an amino acid substitution of anative lysine residue at positions 114 by a tryptophan residue or aglutamic acid with respect to the amino acid sequence of hTlc (SEQ IDNO: 1).

In some embodiments, a lipocalin mutein according to the disclosure mayinclude one or more amino acid mutated to an asparagine residue tointroduce one or more glycosylation sites. In some preferredembodiments, a mutein according to the disclosure includes an amino acidmutation at position 12 of the linear polypeptide sequence of maturehTlc (SEQ ID NO: 1). For example, a mutein according to the disclosuremay have the following mutated amino acid residue with respect to theamino acid sequence of hTlc (SEQ ID NO: 1): Asp 12→Asn.

In some embodiments, a mutein according to the disclosure includes anamino acid substitution at position 5 of the linear polypeptide sequenceof mature hTlc (SEQ ID NO: 1). For example, a mutein according to thedisclosure may have the following mutated amino acid residue withrespect to the amino acid sequence of hTlc (SEQ ID NO: 1): Ala 5→Thr.

Further, in some embodiments, a mutein according to the disclosure mayinclude at least one amino acid substitution of a native negativelycharged residue by neutral residue, wherein the native negativelycharged residue is not involved in binding to LAG-3, and wherein thesubstitution results in an increased isoelectric point (pi) of themutein. In some particular embodiments, such native negatively chargedresidues and positions include Asp 7, Glu 9, Asp 12, Glu 45, Asp 72, Glu73, Asp 80, and Asp 95 with respect to the amino acid sequence of hTlc(SEQ ID NO: 1). In some particular embodiments, such neutral amino acidresidues include Asn, Arg, and Lys. In some further particularembodiments, a mutein according to the disclosure includes one or moreof the following mutated amino acid residues at position 7, 9, 12, 45,72, 73, 80, and 95 of the linear polypeptide sequence of mature hTlc(SEQ ID NO: 1): Asp 7→Asn, Arg, or Lys; Glu 9→Gln, Arg, or Lys; Asp12→Asn or Arg; Glu 45→Arg; Asp 72→Asn, Arg, or Lys; Glu 73→Arg; Asp80→Gly; and Asp 95→Asn, Arg, or Lys. Exemplary muteins include SEQ IDNOs: 57-60, 63, 66.

In some embodiments, a LAG-3-binding hTlc mutein according to thedisclosure includes, at one or more positions corresponding to positions5, 7-8, 10, 14, 16, 25-34, 44, 46, 52-53, 55-56, 58, 60-61, 63, 65-66,69-70, 73, 79-80, 84-86, 89-90, 93, 96-98, 101, 105-106, 108, 110-114,121, 124, 148-150, and 152-154 of the linear polypeptide sequence ofmature hTlc (SEQ ID NO: 1), one or more of the following mutated aminoacid residues: Ala 5→Thr; Asp 7→Gly; Glu 8→Gln; Ile 10→Phe; Ser 14→Pro;Thr 16→Met; Asp 25→Ser; Arg 26→Ser, Asp, Glu, Ala, or Gly; Glu 27→Asp;Phe 28→Cys or Asp; Pro 29→Phe; Glu 30→Trp; Met 31→Ile or Leu; Asn32→Asp, Met, or Thr; Leu 33→Asp; Glu 34→Val; Leu 44→His; Gly 46→Asp; Lys52→Arg; Val 53→Ala; Met 55→Val; Leu 56→Asp; Ser 58→Phe or Asp; Arg60→Phe; Cys 61→Trp; Glu 63→Asp; Lys 65→Glu; Ala 66→Asn; Glu 69→Gly; Lys70→Arg; Glu 73→Ala; Ala 79→Thr or Glu; Asp 80→Gly; His 84→Tyr or Leu;Val 85→Ala or Asp; Ala 86→Asp; Ile 89→Ser or Asn; Arg 90→Ser; Val93→Glu; His 96→Asn; Tyr 97→His; Ile 98→Val; Cys 101→Ser or Phe; Leu105→Cys or Gly; His 106→Ala, Gln, Glu, Lys, or Pro; Lys 108→Tyr or Thr;Val 110→Gly or Asn; Arg 111→Pro; Gly 112→Met, Val, or Leu; Val 113→Alaor Leu; Lys 114→Trp or Ala; Lys 121→Thr; Leu 124→Gln; Arg 148→Trp; Gln149→Leu; Ser 150→Gly; Thr 152→Pro; Cys 153→Ser; and Ser 154→Ala. In someembodiments, a hTlc mutein according to the disclosure includes two ormore, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or even more such as 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or even moremutated amino acid residues at these sequence positions of mature hTlc(SEQ ID NO: 1).

In some embodiments, a LAG-3-binding hTlc mutein according to thedisclosure includes, at one or more positions corresponding to positions14, 25-26, 28, 31-32, 52, 55, 58, 66, 79, 84, 86, 101, 105-106, 108,110, 112-114, and 121 of the linear polypeptide sequence of mature hTlc(SEQ ID NO: 1), one or more of the following mutated amino acidresidues: Ser 14→Pro; Asp 25→Ser; Arg 26→Ser, Asp, Glu, Ala, or Gly; Phe28→Asp; Met 31→Leu; Asn 32→Met or Thr; Lys 52→Arg; Met 55→Val; Ser58→Asp; Ala 66→Asn; Ala 79→Glu; His 84→Tyr or Leu; Ala 86→Asp; Cys101→Phe; Leu 105→Gly; His 106→Gln, Glu, Lys, or Pro; Lys 108→Thr; Val110→Gly or Asn; Gly 112→Met, Val, or Leu; Val 113→Ala or Leu; Lys114→Ala; Lys 121→Thr. In some embodiments, a hTlc mutein according tothe disclosure includes two or more, such as 3, 4, 5, 6, 7, 8, 9, 10,11, 12, or even more such as 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22mutated amino acid residues at these sequence positions of mature hTlc(SEQ ID NO: 1).

In some embodiments, a LAG-3-binding hTlc mutein according to thedisclosure includes, at one or more positions corresponding to positions5, 7-8, 10, 16, 26-34, 44, 46, 53, 56, 58, 60-61, 63, 65-66, 69-70, 73,79-80, 85, 89-90, 93, 96-98, 101, 105-106, 108, 110-111, 114, 121, 124,148-150, and 152-154 of the linear polypeptide sequence of mature hTlc(SEQ ID NO: 1), one or more of the following mutated amino acidresidues: Ala 5→Thr; Asp 7→Gly; Glu 8→Gln; Ile 10→Phe; Thr 16→Met; Arg26→Ser; Glu 27→Asp; Phe 28→Cys; Pro 2→Phe; Glu 30→Trp; Met 31→Ile; Asn32→Asp; Leu 33→Asp; Glu 34→Val; Leu 44→His; Gly 46→Asp; Val 53→Ala; Leu56→Asp; Ser 58→Phe; Arg 60→Phe; Cys 61→Trp; Glu 63→Asp; Lys 65→Glu; Glu69→Gly; Lys 70→Arg; Glu 73→Ala; Ala 79→Thr; Asp 80→Gly; Val 85→Ala orAsp; Ile 89→Ser or Asn; Arg 90→Ser; Val 93→Glu; His 96→Asn; Tyr 97→His;Ile 98→Val; Cys 101→Ser; Leu 105→Cys; His 106→Ala; Lys 108→Tyr; Arg111→Pro; Lys 114→Trp; Leu 124→Gln; Arg 148→Trp; Gln 149→Leu; Ser150→Gly; Thr 152→Pro; Cys 153→Ser; and Ser 154→Ala. In some embodiments,a hTlc mutein according to the disclosure includes two or more, such as3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or even more such as 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or even more mutated aminoacid residues at these sequence positions of mature hTlc (SEQ ID NO: 1).

In some embodiments, a LAG-3-binding hTlc mutein according to thedisclosure includes, at one or more positions corresponding to positions5, 7-8, 10, 16, 26-34, 44, 46, 53, 56, 58, 60-61, 63, 65, 69-70, 73,79-80, 85, 89-90, 93, 96-98, 101, 105-106, 108, 111, 114, 124, 148-150,and 152-154 of the linear polypeptide sequence of mature hTlc (SEQ IDNO: 1), one or more of the following mutated amino acid residues: Ala5→Thr; Asp 7→Gly; Glu 8→Gln; Ile 10→Phe; Thr 16→Met; Arg 26→Ser; Glu27→Asp; Phe 28→Cys; Pro 29→Phe; Glu 30→Trp; Met 31→Ile; Asn 32→Asp; Leu33→Asp; Glu 34→Val; Leu 44→His; Gly 46→Asp; Val 53→Ala; Leu 56→Asp; Ser58→Phe; Arg 60→Phe; Cys 61→Trp; Glu 63→Asp; Lys 65→Glu; Glu 69→Gly; Lys70→Arg; Glu 73→Ala; Ala 79→Thr; Asp 80→Gly; Val 85→Ala or Asp; Ile89→Ser or Asn; Arg 90→Ser; Val 93→Glu; His 96→Asn; Tyr 97→His; Ile98→Val; Cys 101→Ser; Leu 105→Cys; His 106→Ala; Lys 108→Tyr; Arg 111→Pro;Lys 114→Trp; Leu 124→Gln; Arg 148→Trp; Gln 149→Leu; Ser 150→Gly; Thr152→Pro; Cys 153→Ser; and Ser 154→Ala. In some embodiments, a hTlcmutein according to the disclosure includes two or more, such as 3, 4,5, 6, 7, 8, 9, 10, 11, 12, or even more such as 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27 or even more mutated amino acidresidues at these sequence positions of mature hTlc (SEQ ID NO: 1).

In some embodiments, the LAG-3 binding hTlc muteins include thefollowing amino acid mutations in comparison with the linear polypeptidesequence of mature hTlc (SEQ ID NO: 1): Arg 26→Ser; Glu 27→Asp; Phe28→Cys; Pro 29→Phe; Glu 30→Trp; Met 31→Ile; Asn 32→Asp; Leu 33→Asp; Glu34→Val; Leu 56→Asp; Ser 58→Phe; Arg 60→Phe; Cys 61→Trp; Cys 101→Ser; Leu105→Cys; His 106→Ala; Lys 108→Tyr; Arg 111→Pro; Lys 114→Trp; Cys153→Ser; and further one or more, including 2, 3, 4, 5, 6, or 7, or evenmore, of the following amino acid mutations: Ala 5→Thr; Asp 7→Gly; Glu8→Gln; Ile 10→Phe; Thr 16→Met; Leu 44→His; Gly 46→Asp; Val 53→Ala; Glu63→Asp; Lys 65→Glu; Glu 69→Gly; Lys 70→Arg; Glu 73→Ala; Ala 79→Thr; Asp80→Gly; Val 85→Ala or Asp; Ile 89→Ser or Asn; Arg 90→Ser; Val 93→Glu;His 96→Asn; Tyr 97→His; Ile 98→Val; Leu 124→Gln; Arg 148→Trp; Gln149→Leu; Ser 150→Gly; Thr 152→Pro; and Ser 154→Ala.

In some embodiments, the LAG-3 binding hTlc muteins include thefollowing amino acid mutations in comparison with the linear polypeptidesequence of mature hTlc (SEQ ID NO: 1): Ser 14→Pro; Asp 25→Ser; Phe28→Asp; Lys 52→Arg; Met 55→Val; Ser 58→Asp; Ala 66→Asn; Ala 79→Glu; Ala86→Asp; Cys 101→Phe; Leu 105→Gly; Lys 108→Thr; Lys 114→Ala; Lys 121→Thr;and one or more, including 2, 3, 4, 5, 6, 7, or even more, of thefollowing amino acid mutations: Arg 26→Ser, Asp, Glu, Ala, or Gly; Met31→Leu; Asn 32→Thr; Leu 56→Asp; His 84→Tyr or Leu; His 106→Gln, Glu,Lys, or Pro; Val 110→Gly or Asn; Gly 112→Met, Val or Leu; Val 113→Ala orLeu.

In some embodiments, the LAG-3 binding hTlc muteins include thefollowing amino acid mutations in comparison with the linear polypeptidesequence of mature hTlc (SEQ ID NO: 1): Ser 14→Pro; Asp 25→Ser; Phe28→Asp; Lys 52→Arg; Met 55→Val; Ser 58→Asp; Ala 66→Asn; Ala 79→Glu; Ala86→Asp; Cys 101→Phe; Leu 105→Gly; Lys 108→Thr; Lys 114→Ala; Lys 121→Thr;and one or more, including 2, 3, 4, 5, 6, 7, or even more, of thefollowing amino acid mutations: Arg 26→Ser, Asp, Glu, Ala, or Gly; Met31→Leu; Asn 32→Thr; His 84→Tyr or Leu; His 106→Gln, Glu, Lys, or Pro;Val 110→Gly or Asn; Gly 112→Met, Val or Leu; Val 113→Ala or Leu.

In some embodiments, the LAG-3 binding hTlc muteins include thefollowing amino acid mutations in comparison with the linear polypeptidesequence of mature hTlc (SEQ ID NO: 1): Ser 14→Pro; Asp 25→Ser; Phe28→Asp; Lys 52→Arg; Met 55→Val; Ser 58→Asp; Ala 66→Asn; Ala 79→Glu; Ala86→Asp; Cys 101→Phe; Leu 105→Gly; Lys 108→Thr; Lys 114→Ala; Lys 121→Thr;and one or more, including 2, 3, 4, 5, 6, 7, or even more, of thefollowing amino acid mutations: Arg 26→Ser, Asp, Glu, or Ala; Met31→Leu; Asn 32→Thr; Leu 56→Asp; His 84→Tyr or Leu; His 106→Glu, Lys, orPro; Val 110→Asn; Gly 112→Val or Leu; Val 113→Ala or Leu.

In some additional embodiments, the LAG-3 binding hTlc muteins includeone of the following sets of amino acid mutations in comparison with thelinear polypeptide sequence of mature hTlc (SEQ ID NO: 1):

-   -   (a) Ala 5→Thr; Glu 8→Gln; Arg 26→Ser; Glu 27→Asp; Phe 28→Cys;        Pro 29→Phe; Glu 30→Trp; Met 31→Ile; Asn 32→Asp; Leu 33→Asp; Glu        34→Val; Leu 56→Asp; Ser 58→Phe; Arg 60→Phe; Cys 61→Trp; Lys        65→Glu; Glu 69→Gly; Val 85→Ala; Cys 101→Ser; Leu 105→Cys; His        106→Ala; Lys 108→Tyr; Arg 111→Pro; Lys 114→Trp; Cys 153→Ser; and        Ser 154→Ala;    -   (b) Ala 5→Thr; Arg 26→Ser; Glu 27→Asp; Phe 28→Cys; Pro 29→Phe;        Glu 30→Trp; Met 31→Ile; Asn 32→Asp; Leu 33→Asp; Glu 34→Val; Gly        46→Asp; Leu 56→Asp; Ser 58→Phe; Arg 60→Phe; Cys 61→Trp; Lys        65→Glu; Val 85→Ala; Cys 101→Ser; Leu 105→Cys; His 106→Ala; Lys        108→Tyr; Arg 111→Pro; Lys 114→Trp; Ser 150→Gly; and Cys 153→Ser;    -   (c) Asp 7→Gly; Arg 26→Ser; Glu 27→Asp; Phe 28→Cys; Pro 29→Phe;        Glu 30→Trp; Met 31→Ile; Asn 32→Asp; Leu 33→Asp; Glu 34→Val; Leu        56→Asp; Ser 58→Phe; Arg 60→Phe; Cys 61→Trp; Val 85→Asp; Cys        101→Ser; Leu 105→Cys; His 106→Ala; Lys 108→Tyr; Arg 111→Pro; Lys        114→Trp; Arg 148→Trp; Thr 152→Pro; and Cys 153→Ser;    -   (d) Ala 5→Thr; Arg 26→Ser; Glu 27→Asp; Phe 28→Cys; Pro 29→Phe;        Glu 30→Trp; Met 31→Ile; Asn 32→Asp; Leu 33→Asp; Glu 34→Val; Val        53→Ala; Leu 56→Asp; Ser 58→Phe; Arg 60→Phe; Cys 61→Trp; Lys        65→Glu; Ala 79→Thr; Tyr 97→His; Cys 101→Ser; Leu 105→Cys; His        106→Ala; Lys 108→Tyr; Arg 111→Pro; Lys 114→Trp; and Cys 153→Ser;    -   (e) Arg 26→Ser; Glu 27→Asp; Phe 28→Cys; Pro 29→Phe; Glu 30→Trp;        Met 31→Ile; Asn 32→Asp; Leu 33→Asp; Glu 34→Val; Leu 56→Asp; Ser        58→Phe; Arg 60→Phe; Cys 61→Trp; Glu 63→Asp; Val 85→Asp; Arg        90→Ser; His 96→Asn; Cys 101→Ser; Leu 105→Cys; His 106→Ala; Lys        108→Tyr; Arg 111→Pro; Lys 114→Trp; Leu 124→Gln; and Cys 153→Ser;    -   (f) Thr 16→Met; Arg 26→Ser; Glu 27→Asp; Phe 28→Cys; Pro 29→Phe;        Glu 30→Trp; Met 31→Ile; Asn 32→Asp; Leu 33→Asp; Glu 34→Val; Leu        44→His; Leu 56→Asp; Ser 58→Phe; Arg 60→Phe; Cys 61→Trp; Lys        65→Glu; Ile 89→Ser; Cys 101→Ser; Leu 105→Cys; His 106→Ala; Lys        108→Tyr; Arg 111→Pro; Lys 114→Trp; and Cys 153→Ser;    -   (g) Arg 26→Ser; Glu 27→Asp; Phe 28→Cys; Pro 29→Phe; Glu 30→Trp;        Met 31→Ile; Asn 32→Asp; Leu 33→Asp; Glu 34→Val; Leu 56→Asp; Ser        58→Phe; Arg 60→Phe; Cys 61→Trp; Glu 63→Asp; Lys 65→Glu; Cys        101→Ser; Leu 105→Cys; His 106→Ala; Lys 108→Tyr; Arg 111→Pro; Lys        114→Trp; Gln 149→Leu; and Cys 153→Ser;    -   (h) Arg 26→Ser; Glu 27→Asp; Phe 28→Cys; Pro 29→Phe; Glu 30→Trp;        Met 31→Ile; Asn 32→Asp; Leu 33→Asp; Glu 34→Val; Leu 56→Asp; Ser        58→Phe; Arg 60→Phe; Cys 61→Trp; Lys 65→Glu; Lys 70→Arg; Cys        101→Ser; Leu 105→Cys; His 106→Ala; Lys 108→Tyr; Arg 111→Pro; Lys        114→Trp; and Cys 153→Ser;    -   (i) Ala 5→Thr; Arg 26→Ser; Glu 27→Asp; Phe 28→Cys; Pro 29→Phe;        Glu 30→Trp; Met 31→Ile; Asn 32→Asp; Leu 33→Asp; Glu 34→Val; Leu        56→Asp; Ser 58→Phe; Arg 60→Phe; Cys 61→Trp; Lys 65→Glu; Asp        80→Gly; Ile 89→Asn; Ile 98→Val; Cys 101→Ser; Leu 105→Cys; His        106→Ala; Lys 108→Tyr; Arg 111→Pro; Lys 114→Trp; and Cys 153→Ser;    -   (j) Ile 10→Phe; Arg 26→Ser; Glu 27→Asp; Phe 28→Cys; Pro 29→Phe;        Glu 30→Trp; Met 31→Ile; Asn 32→Asp; Leu 33→Asp; Glu 34→Val; Leu        56→Asp; Ser 58→Phe; Arg 60→Phe; Cys 61→Trp; Lys 65→Glu; Glu        73→Ala; Ile 89→Asn; Val 93→Glu; Cys 101→Ser; Leu 105→Cys; His        106→Ala; Lys 108→Tyr; Arg 111→Pro; Lys 114→Trp; and Cys 153→Ser;    -   (k) Ala 5→Thr; Glu 8→Gln; Arg 26→Ser; Glu 27→Asp; Phe 28→Cys;        Pro 29→

Phe; Glu 30→Trp; Met 31→Ile; Asn 32→Asp; Leu 33→Asp; Glu 34→Val; Leu56→Asp; Ser 58→Phe; Arg 60→Phe; Cys 61→Trp; Lys 65→Glu; Glu 69→Gly; Val85→Ala; Cys 101→Ser; Leu 105→Cys; His 106→Ala; Lys 108→Tyr; Arg 111→Pro;Lys 114→Trp; Cys 153→Ser; and Ser 154→Ala;

-   -   (l) Arg 26→Ser; Glu 27→Asp; Phe 28→Cys; Pro 29→Phe; Glu 30→Trp;        Met 31→Ile; Asn 32→Asp; Leu 33→Asp; Glu 34→Val; Leu 56→Asp; Ser        58→Phe; Arg 60→Phe; Cys 61→Trp; Cys 101→Ser; Leu 105→Cys; His        106→Ala; Lys 108→Tyr; Arg 111→Pro; Lys 114→Trp; and Cys 153→Ser;    -   (m) Ser 14→Pro; Asp 25→Ser; Arg 26→Asp; Phe 28→Asp; Asn 32→Thr;        Lys 52→Arg; Met 55→Val; Ser 58→Asp; Ala 66→Asn; Ala 79→Glu; His        84→Tyr; Ala 86→Asp; Cys 101→Phe; Leu 105→Gly; Lys 108→Thr; Val        110→Gly; Gly 112→Met; Lys 114→Ala; and Lys 121→Thr;    -   (n) Ser 14→Pro; Asp 25→Ser; Arg 26→Glu; Phe 28→Asp; Met 31→Leu;        Asn 32→Thr; Lys 52→Arg; Met 55→Val; Ser 58→Asp; Ala 66→Asn; Ala        79→Glu; His 84→Tyr; Ala 86→Asp; Cys 101→Phe; Leu 105→Gly; His        106→Gln; Lys 108→Thr; Val 110→Gly; Gly 112→Met; Lys 114→Ala; and        Lys 121→Thr;    -   (o) Ser 14→Pro; Asp 25→Ser; Arg 26→Glu; Phe 28→Asp; Asn 32→Thr;        Lys 52→Arg; Met 55→Val; Ser 58→Asp; Ala 66→Asn; Ala 79→Glu; His        84→Tyr; Ala 86→Asp; Cys 101→Phe; Leu 105→Gly; His 106→Glu; Lys        108→Thr; Val 110→Gly; Gly 112→Val; Lys 114→Ala; and Lys 121→Thr;    -   (p) Ser 14→Pro; Asp 25→Ser; Arg 26→Asp; Phe 28→Asp; Asn 32→Thr;        Lys 52→Arg; Met 55→Val; Ser 58→Asp; Ala 66→Asn; Ala 79→Glu; His        84→Tyr; Ala 86→Asp; Cys 101→Phe; Leu 105→Gly; His 106→Gln; Lys        108→Thr; Val 110→Gly; Gly 112→Leu; Lys 114→Ala; and Lys 121→Thr;    -   (q) Ser 14→Pro; Asp 25→Ser; Arg 26→Ser; Phe 28→Asp; Asn 32→Thr;        Lys 52→Arg; Met 55→Val; Ser 58→Asp; Ala 66→Asn; Ala 79→Glu; His        84→Tyr; Ala 86→Asp; Cys 101→Phe; Leu 105→Gly; His 106→Gln; Lys        108→Thr; Val 110→Gly; Gly 112→Met; Lys 114→Ala; and Lys 121→Thr;    -   (r) Ser 14→Pro; Asp 25→Ser; Arg 26→Ala; Phe 28→Asp; Asn 32→Thr;        Lys 52→Arg; Met 55→Val; Ser 58→Asp; Ala 66→Asn; Ala 79→Glu; His        84→Tyr; Ala 86→Asp; Cys 101→Phe; Leu 105→Gly; His 106→Lys; Lys        108→Thr; Val 110→Gly; Gly 112→Met; Lys 114→Ala; and Lys 121→Thr;    -   (s) Ser 14→Pro; Asp 25→Ser; Phe 28→Asp; Asn 32→Thr; Lys 52→Arg;        Met 55→Val; Ser 58→Asp; Ala 66→Asn; Ala 79→Glu; Ala 86→Asp; Cys        101→Phe; Leu 105→Gly; His 106→Gln; Lys 108→Thr; Val 110→Asn; Gly        112→Met; Val 113→Ala; Lys 114→Ala; and Lys 121→Thr;    -   (t) Ser 14→Pro; Asp 25→Ser; Arg 26→Gly; Phe 28→Asp; Met 31→Leu;        Asn 32→Thr; Lys 52→Arg; Met 55→Val; Ser 58→Asp; Ala 66→Asn; Ala        79→Glu; His 84→Tyr; Ala 86→Asp; Cys 101→Phe; Leu 105→Gly; His        106→Pro; Lys 108→Thr; Val 110→Gly; Gly 112→Met; Lys 114→Ala; and        Lys 121→Thr;    -   (u) Ser 14→Pro; Asp 25→Ser; Arg 26→Asp; Phe 28→Asp; Asn 32→Thr;        Lys 52→Arg; Met 55→Val; Ser 58→Asp; Ala 66→Asn; Ala 79→Glu; His        84→Leu; Ala 86→Asp; Cys 101→Phe; Leu 105→Gly; His 106→Gln; Lys        108→Thr; Val 110→Gly; Gly 112→Met; Val 113→Leu; Lys 114→Ala; and        Lys 121→Thr;    -   (v) Ser 14→Pro; Asp 25→Ser; Arg 26→Gly; Phe 28→Asp; Asn 32→Met;        Lys 52→Arg; Met 55→Val; Ser 58→Asp; Ala 66→Asn; Ala 79→Glu; Ala        86→Asp; Cys 101→Phe; Leu 105→Gly; His 106→Gln; Lys 108→Thr; Val        110→Gly; Gly 112→Met; Lys 114→Ala; and Lys 121→Thr; or    -   (w) Arg 26→Ser; Glu 27→Asp; Phe 28→Cys; Pro 29→Phe; Glu 30→Trp;        Met 31→Ile; Asn 32→Asp; Leu 33→Asp; Glu 34→Val; Leu 56→Asp; Ser        58→Phe; Arg 60→Phe; Glu 63→Asp; Lys 65→Glu; Cys 101→Ser; Leu        105→Cys; His 106→Ala; Lys 108→Tyr; Arg 111→Pro; Lys 114→Trp; and        Gln 149→Leu.

In some additional embodiments, the LAG-3 binding hTlc muteins includeone of the following sets of amino acid mutations in comparison with thelinear polypeptide sequence of mature hTlc (SEQ ID NO: 1):

-   -   (a) Ala 5→Thr; Glu 8→Gln; Lys 65→Glu; Glu 69→Gly; Val 85→Ala;        and Ser 154→Ala;    -   (b) Ala 5→Thr; Gly 46→Asp; Lys 65→Glu; Val 85→Ala; and Ser        150→Gly    -   (c) Asp 7→Gly; Val 85→Asp; Arg 148→Trp; and Thr 152→Pro;    -   (d) Ala 5→Thr; Val 53→Ala; Lys 65→Glu; Ala 79→Thr; and Tyr        97→His;    -   (e) Glu 63→Asp; Val 85→Asp; Arg 90→Ser; His 96→Asn; and Leu        124→Gln;    -   (f) Thr 16→Met; Leu 44→His; Lys 65→Glu; and Ile 89→Ser;    -   (g) Glu 63→Asp; Lys 65→Glu; and Gln 149→Leu;    -   (h) Lys 65→Glu and Lys 70→Arg;    -   (i) Ala 5→Thr; Lys 65→Glu; Asp 80→Gly; Ile 89→Asn; and Ile        98→Val    -   (j) Ile 10→Phe; Lys 65→Glu; Glu 73→Ala; Ile 89→Asn; and Val        93→Glu;    -   (k) Arg 26→Asp; Asn 32→Thr; His 84→Tyr; Val 110→Gly; and Gly        112→Met;    -   (l) Arg 26→Glu; Met 31→Leu; Asn 32→Thr; His 84→Tyr; His 106→Gln;        Val 11 →Gly; and Gly 112→Met;    -   (m) Arg 26→Glu; Asn 32→Thr; His 84→Tyr; His 106→Glu; and Gly        112→Val;    -   (n) Arg 26→Asp; Asn 32→Thr; His 84→Tyr; His 106→Gln; Val        110→Gly; and Gly 112→Leu;    -   (o) Arg 26→Ser; Asn 32→Thr; His 84→Tyr; His 106→Gln; Val        110→Gly; and Gly 112→Met;    -   (p) Arg 26→Ala; Asn 32→Thr; His 84→Tyr; His 106→Lys; Val        110→Gly; and Gly 112→Met;    -   (q) Asn 32→Thr; His 106→Gln; Val 110→Asn; Gly 112→Met; and Val        113→Ala;    -   (r) Arg 26→Gly; Met 31→Leu; Asn 32→Thr; His 84→Tyr; His 106→Pro;        Val 110→Gly; and Gly 112→Met; or    -   (s) Arg 26→Asp; Asn 32→Thr; His 84→Leu; His 106→Gln; Val        110→Gly; Gly 112→Met; and Val 113→Leu.

In some additional embodiments the LAG-3 binding hTlc mutein includesthe following amino acid mutation in comparison with the linearpolypeptide sequence of the hTlc (SEQ ID NO: 1): insertion of Probetween positions 156 and 157.

In the residual region, i.e., the region differing from sequencepositions 5, 7-8, 10, 14, 16, 25-34, 44, 46, 52-53, 55-56, 58, 60-61,63, 65-66, 69-70, 73, 79-80, 84-86, 89-90, 93, 96-98, 101, 105-106, 108,110-114, 121, 124, 148-150, 152-154, and 157, a hTlc mutein of thedisclosure may include the wild-type (natural) amino acid sequence ofmature hTlc (SEQ ID NO: 1) outside the mutated amino acid sequencepositions or mutated amino acid residues at such positions.

Unless otherwise indicated, the position of a residue of a hTlc muteindescribed herein is numbered in comparison with the linear polypeptidesequence of the hTlc (SEQ ID NO: 1).

In still further embodiments, a hTlc mutein according to the currentdisclosure has at least 70% sequence identity or at least 70% sequencehomology to the sequence of mature hTlc (SEQ ID NO: 1). As anillustrative example, the mutein of the SEQ ID NO: 8 has an amino acidsequence identity or a sequence homology of approximately 81.8% with theamino acid sequence of mature hTlc (SEQ ID NO: 1).

In further particular embodiments, a hTlc mutein of the disclosurecomprises an amino acid sequence as set forth in any one of SEQ ID NOs:7-28, 57-70, and 85-95 or a fragment or variant thereof.

In further particular embodiments, a hTlc mutein of the disclosure hasat least 75%, at least 80%, at least 85% or higher, at least 90% orhigher, at least 95% or higher, at least 97.5% or higher, at least 98%or higher or at least 99% or higher sequence identity to an amino acidsequence selected from the group consisting of SEQ ID NOs: 8-18, 20-28,57-70 and 85-95.

The disclosure also includes structural homologues of a hTlc muteinhaving an amino acid sequence selected from the group consisting of SEQID NOs: 7-28, 57-70, and 85-95, which structural homologues have anamino acid sequence homology or sequence identity of more than about60%, preferably more than 65%, more than 70%, more than 75%, more than80%, more than 85%, more than 90%, more than 92%, and most preferablymore than 95% in relation to said hTlc mutein.

A hTlc mutein according to the present disclosure can be obtained bymeans of mutagenesis of a naturally occurring form of mature hTlc (SEQID NO: 1). In some embodiments of the mutagenesis, a substitution (orreplacement) is a conservative substitution. Nevertheless, anysubstitution—including non-conservative substitution or one or more fromthe exemplary substitutions below—is envisaged as long as the lipocalinmutein retains its capability to bind to LAG-3, and/or it has a sequenceidentity to the then substituted sequence in that it is at least 60%,such as at least 65%, at least 70%, at least 75%, at least 80%, at least85% or higher sequence identity to the amino acid sequence of maturehTlc (SEQ ID NO: 1).

In some particular embodiments, the present disclosure provides alipocalin mutein that binds human LAG-3 with an affinity measured by aK_(d) of about 10 nM or lower, 5 nM or lower, 4 nM or lower, 3 nM orlower, 2 nM or lower, 1 nM or lower, 0.5 nM or lower, 0.1 nM or lower or0.05 nM or lower. In some embodiments, the lipocalin mutein has at least90% or higher, such as 95% or higher, 97.5% or higher, 98% or higher, or99% or higher sequence identity to the amino acid sequence of any one ofSEQ ID NOs: 7 and 19.

2. Applications of Lipocalin Muteins Specific for LAG-3

Numerous possible applications for the LAG-3-binding lipocalin muteinsof the disclosure exist in medicine.

In one further aspect, the disclosure relates to the use of aLAG-3-binding lipocalin mutein disclosed herein for detecting LAG-3 in asample as well as a respective method of diagnosis.

The present disclosure also involves the use of one or moreLAG-3-binding lipocalin muteins as described for complex formation withLAG-3.

Therefore, in another aspect of the disclosure, the disclosed lipocalinmuteins are used for the detection of LAG-3. Such use may include thesteps of contacting one or more said muteins, under suitable conditions,with a sample suspected of containing LAG-3, thereby allowing theformation of a complex between the muteins and LAG-3, and detecting thecomplex by a suitable signal. The detectable signal can be caused by alabel, as explained above, or by a change of physical properties due tothe binding, i.e., the complex formation, itself. One example is surfaceplasmon resonance, the value of which is changed during binding ofbinding partners from which one is immobilized on a surface such as agold foil.

The LAG-3-binding lipocalin muteins disclosed herein may also be usedfor the separation of LAG-3. Such use may include the steps ofcontacting one or more said muteins, under suitable conditions, with asample supposed to contain LAG-3, thereby allowing the formation of acomplex between the muteins and LAG-3 and separating the complex fromthe sample.

In the use of the disclosed muteins for the detection of LAG-3 as wellas the separation of LAG-3, the muteins and/or LAG-3 or a domain orfragment thereof may be immobilized on a suitable solid phase.

In still another aspect, the present disclosure features a diagnostic oranalytical kit comprising a LAG-3-binding lipocalin mutein according tothe disclosure.

In addition to their use in diagnostics, in yet another aspect, thedisclosure contemplates a pharmaceutical composition comprising a muteinof the disclosure and a pharmaceutically acceptable excipient.

Furthermore, the present disclosure provides human lipocalin muteinsthat bind LAG-3 for use as anti-cancer agents and/or immune modulators.As such the lipocalin muteins of the present disclosure that bind LAG-3are envisaged to be used in a method of treatment or prevention of humandiseases such as cancer, infectious diseases, and autoimmune diseases.Accordingly, also provided are methods of treatment or prevention ofhuman diseases such as cancer, infectious diseases, and autoimmunediseases in a subject in need thereof, comprising administering to saidsubject a therapeutically effective amount of a lipocalin mutein of thepresent invention that binds LAG-3.

B. Lipocalin Muteins of the Disclosure

Lipocalins are proteinaceous binding molecules that have naturallyevolved to bind ligands. Lipocalins occur in many organisms, includingvertebrates, insects, plants and bacteria. The members of the lipocalinprotein family (Pervaiz and Brew, FASEB J, 1987) are typically smallsecreted proteins and have a single polypeptide chain. They arecharacterized by a range of different molecular-recognition properties:their binding to various, principally hydrophobic, small molecules (suchas retinoids, fatty acids, cholesterols, prostaglandins, biliverdins,pheromones, tastants, and odorants), and to specific cell-surfacereceptors and their formation of macromolecular complexes. Although theyhave, in the past, been classified primarily as transport proteins, itis now clear that the lipocalins fulfill a variety of physiologicalfunctions. These include roles in retinol transport, olfaction,pheromone signaling, and the synthesis of prostaglandins. Lipocalinshave also been implicated in the regulation of the immune response andthe mediation of cell homoeostasis (reviewed, e.g., in Flower et al.,Biochim Biophys Acta, 2000, Flower, Biochem J, 1996).

Lipocalins share unusually low levels of overall sequence conservation,often with sequence identities of less than 20%. In strong contrast,their overall folding pattern is highly conserved. The central part ofthe lipocalin structure consists of a single eight-strandedanti-parallel p-sheet, closed back on itself to form a continuouslyhydrogen-bonded β-barrel. This β-barrel forms a central cavity. One endof the barrel is sterically blocked by the N-terminal peptide segmentthat runs across its bottom as well as three peptide loops connectingthe β-strands. The other end of the β-barrel is open to the solvent andencompasses a target-binding site, formed by four flexible peptideloops. It is the diversity of the loops in the otherwise rigid lipocalinscaffold that gives rise to a variety of different binding modes, eachcapable of accommodating targets of different size, shape, and chemicalcharacter (reviewed, e.g., in Skerra, Biochim Biophys Acta, 2000, Floweret al., Biochim Biophys Acta, 2000, Flower, Biochem J, 1996).

When used herein in the context of the lipocalin muteins of the presentdisclosure that bind to LAG-3, the term “specific for” includes that thelipocalin mutein is directed against, binds to, or reacts with LAG-3.Thus, being directed to, binding to or reacting with includes that thelipocalin mutein specifically binds to LAG-3. The term “specifically” inthis context means that the lipocalin mutein reacts with a LAG-3protein, as described herein, but essentially not with another protein.The term “another protein” includes any non-LAG-3 protein, includingproteins closely related to or being homologous to LAG-3 against whichthe lipocalins disclosed herein are directed to. However, LAG-3proteins, fragments and/or variants from species other than human suchas those described in the context of the definition “subject” are notexcluded by the term “another protein.” The term “does not essentiallybind” means that the lipocalin mutein of the present disclosure does notbind another protein, i.e., shows a cross-reactivity of less than 30%,preferably 20%, more preferably 10%, particularly preferably less than9, 8, 7, 6 or 5%. Whether the lipocalin specifically reacts as definedherein above can easily be tested, inter alia, by comparing the reactionof a lipocalin mutein of the present disclosure with LAG-3 and thereaction of said lipocalin with (an)other protein(s). “Specific binding”can also be determined, for example, in accordance with Western blot,ELISA, RIA, ECL, IRMA, FACS, IHC, and peptide scans.

The amino acid sequence of a lipocalin mutein according to thedisclosure has a high sequence identity to the reference lipocalin, forexample hTlc, as compared to such mutein's sequence identity withanother lipocalin. In this general context, the amino acid sequence of alipocalin mutein of the combination according to the disclosure is atleast substantially similar to the amino acid sequence of thecorresponding wild-type or reference lipocalin. A respective sequence ofa lipocalin mutein of the combination according to the disclosure, beingsubstantially similar to the sequences of the corresponding referencelipocalin, has at least 65%, at least 70%, at least 75%, at least 80%,at least 82%, at least 85%, at least 87%, at least 90% identity,including at least 95% identity to the sequence of the correspondinglipocalin. In this regard, a lipocalin mutein of the disclosure ofcourse may contain, in comparison substitutions as described hereinwhich renders the lipocalin mutein capable of binding to LAG-3.Typically, a mutein of a lipocalin includes one or moremutations—relative to the sequence of the reference lipocalin—of aminoacids in the four loops at the open end of the ligand binding site oflipocalins (cf. above). As explained above, these regions are essentialin determining the binding specificity of a lipocalin mutein for adesired target.

A mutein of the present disclosure may also contain mutations in regionsoutside of the four flexible peptide loops that form the target bindingsite of the lipocalin. For example, a mutein of the present inventionmay contain one or more mutations in one or more of the three peptideloops (designated BC, DE, and FG) connecting the 8-strands at the closedend of the lipocalin. As an illustrative example, a mutein derived froma polypeptide of tear lipocalin or a homologue thereof, may have 1, 2,3, 4 or more mutated amino acid residues at any sequence position in theN-terminal region and/or in the three peptide loops BC, DE, and FGarranged at the end of the β-barrel structure that is located oppositeto the natural lipocalin binding pocket. As a further illustrativeexample, a mutein derived from a polypeptide of tear lipocalin or ahomologue thereof, may have no mutated amino acid residues in peptideloop DE arranged at the end of the β-barrel structure, compared towild-type sequence of tear lipocalin.

A lipocalin mutein according to the disclosure includes one or more,such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,or even 20 substitutions in comparison to the corresponding nativelipocalin, provided that such a lipocalin mutein should be capable ofbinding to LAG-3. For example, a lipocalin mutein can have asubstitution at a position corresponding to a distinct position (i.e.,at a corresponding position) of the wild-type lipocalin having thewild-type sequence of, for example, hTlc. In some embodiments, alipocalin mutein of the combination according to the disclosure includesat least two amino acid substitutions, including 2, 3, 4, 5, or evenmore, amino acid substitutions of a native amino acid by an arginineresidue. Accordingly, the nucleic acid of a “reference protein” scaffoldas described herein is subject to mutagenesis with the aim of generatinga lipocalin mutein which is capable of binding to LAG-3.

Also, a lipocalin mutein of the present disclosure can comprise aheterologous amino acid sequence, such as a Strep-tag II sequence, atits N-or C-Terminus, preferably C-terminus, such as in SEQ ID NO: 53 andSEQ ID NO: 54, without affecting the biological activity (binding to itstarget, e.g. LAG-3) of the lipocalin mutein.

Likewise, a lipocalin mutein of the present disclosure may lack 1, 2, 3,4, or more amino acids at its N-terminal end and/or 1, 2, or more aminoacids at its C-terminal end, in comparison to the respective wild-typetear lipocalin; for example, SEQ ID NOs: 7-28, 57-70, and 85-95.

In some embodiments, a substitution (or replacement) is a conservativesubstitution. Nevertheless, any substitution—including non-conservativesubstitution or one or more of the exemplary substitutions listedbelow—is envisaged as long as the lipocalin mutein retains itscapability to bind to LAG-3, and/or it has an identity to the thensubstituted sequence in that it is at least 60%, such as at least 65%,at least 70%, at least 75%, at least 80%, at least 85% or higheridentical to the “reference sequence”.

Conservative substitutions are generally the following substitutions,listed according to the amino acid to be mutated, each followed by oneor more replacement(s) that can be taken to be conservative: Ala→Gly,Ser, Val; Arg→Lys; Asn→Gln, His; Asp→Glu; Cys→Ser; Gln→Asn; Glu→Asp;Gly→Ala; His→Arg, Asn, Gln; Ile→Leu, Val; Leu→Ile, Val; Lys→Arg, Gln,Glu; Met→Leu, Tyr, Ile; Phe→Met, Leu, Tyr; Ser→Thr; Thr→Ser; Trp→Tyr;Tyr→Trp, Phe; Val→Ile, Leu. Other substitutions are also permissible andcan be determined empirically or in accord with other known conservativeor non-conservative substitutions. As a further orientation, thefollowing eight groups each contain amino acids that can typically betaken to define conservative substitutions for one another:

-   a. Alanine (Ala), Glycine (Gly);-   b. Aspartic acid (Asp), Glutamic acid (Glu);-   c. Asparagine (Asn), Glutamine (Gin);-   d. Arginine (Arg), Lysine (Lys);-   e. Isoleucine (Ile), Leucine (Leu), Methionine (Met), Valine (Val);-   f. Phenylalanine (Phe), Tyrosine (Tyr), Tryptophan (Trp);-   g. Serine (Ser), Threonine (Thr); and-   h. Cysteine (Cys), Methionine (Met)

If such substitutions result in a change in biological activity, thenmore substantial changes, such as the following, or as further describedbelow in reference to amino acid classes, may be introduced and theproducts screened for a desired characteristic. Examples of such moresubstantial changes are: Ala→Leu, Ile; Arg→Gln; Asn→Asp, Lys, Arg, His;Asp→Asn; Cys→Ala; Gln→Glu; Glu→Gln; His→Lys; Ile→Met, Ala, Phe; Leu→Ala,Met, Norleucine; Lys→Asn; Met→Phe; Phe→Val, Ile, Ala; Trp→Phe; Tyr→Thr,Ser; Val→Met, Phe, Ala.

Substantial modifications in the biological properties of the lipocalinare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties: (1) hydrophobic: norleucine, methionine, alanine, valine,leucine, iso-leucine; (2) neutral hydrophilic: cysteine, serine,threonine; (3) acidic: aspartic acid, glutamic acid; (4) basic:asparagine, glutamine, histidine, lysine, arginine; (5) residues thatinfluence chain orientation: glycine, proline; and (6) aromatic:tryptophan, tyrosine, phenylalanine.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Any cysteine residue not involved inmaintaining the proper conformation of the respective lipocalin also maybe substituted, generally with serine, to improve the oxidativestability of the molecule and prevent aberrant crosslinking. Conversely,cysteine bond(s) may be added to the lipocalin to improve its stability.

Any mutation, including an insertion as discussed above, can beaccomplished very easily on the nucleic acid, e.g., DNA level usingestablished standard methods. Illustrative examples of alterations ofthe amino acid sequence are insertions or deletions as well as aminoacid substitutions. Such substitutions may be conservative, i.e., anamino acid residue is replaced with an amino acid residue of chemicallysimilar properties, in particular with regard to polarity as well assize. Examples of conservative substitutions are the replacements amongthe members of the following groups: 1) alanine, serine, and threonine;2) aspartic acid and glutamic acid; 3) asparagine and glutamine; 4)arginine and lysine; 5) iso-leucine, leucine, methionine, and valine;and 6) phenylalanine, tyrosine, and tryptophan. On the other hand, it isalso possible to introduce non-conservative alterations in the aminoacid sequence. In addition, instead of replacing single amino acidresidues, it is also possible to either insert or delete one or morecontinuous amino acids of the primary structure of tear lipocalin aslong as these deletions or insertion result in a stablefolded/functional mutein.

Modifications of the amino acid sequence include directed mutagenesis ofsingle amino acid positions in order to simplify sub-cloning of themutated lipocalin gene or its parts by incorporating cleavage sites forcertain restriction enzymes. In addition, these mutations can also beincorporated to further improve the affinity of a lipocalin mutein for agiven target such as LAG-3. Furthermore, mutations can be introduced inorder to modulate certain characteristics of the mutein such as toimprove folding stability, serum stability, protein resistance or watersolubility or to reduce aggregation tendency, if necessary. For example,naturally occurring cysteine residues may be mutated to other aminoacids to prevent disulphide bridge formation. It is also possible todeliberately mutate other amino acid sequence positions to cysteine inorder to introduce new reactive groups, for example for the conjugationto other compounds, such as polyethylene glycol (PEG), hydroxyethylstarch (HES), biotin, peptides or proteins, or for the formation ofnon-naturally occurring disulphide linkages. The generated thiol moietymay be used to PEGylate or HESylate the mutein, for example, in order toincrease the serum half-life of a respective lipocalin mutein. Exemplarypossibilities of such a mutation to introduce a cysteine residue intothe amino acid sequence of a hTlc mutein include the substitutions Thr40→Cys, Glu 73→Cys, Arg 90→Cys, Asp 95→Cys, and Glu 131→Cys. Thegenerated thiol moiety at the side of any of the amino acid positions40, 73, 90, 95, and/or 131 may be used to PEGylate or HESylate themutein, for example, in order to increase the serum half-life of arespective hTlc mutein.

In some embodiments, if one of the above moieties is conjugated to alipocalin mutein of the disclosure, conjugation to an amino acid sidechain can be advantageous. Suitable amino acid side chains may occurnaturally in the amino acid sequence of a human lipocalin or may beintroduced by mutagenesis. In case a suitable binding site is introducedvia mutagenesis, one possibility is the replacement of an amino acid atthe appropriate position by a cysteine residue. For example, suchmutation includes at least one of Thr 40→Cys, Glu 73→Cys, Arg 90'Cys,Asp 95'Cys or Glu 131→Cys substitution in the wild-type sequence ofhuman tear lipocalin. The newly created cysteine residue at any of thesepositions can then be utilized to conjugate the mutein to moietyprolonging the serum half-life of the mutein, such as PEG or anactivated derivative thereof.

In another embodiment, in order to provide suitable amino acid sidechains for conjugating one of the above compounds to a lipocalin muteinaccording to the present disclosure, artificial amino acids may beintroduced by mutagenesis. Generally, such artificial amino acids aredesigned to be more reactive and thus to facilitate the conjugation tothe desired compound. One example of such an artificial amino acid thatmay be introduced via an artificial tRNA is para-acetyl-phenylalanine.

For several applications of the muteins disclosed herein it may beadvantageous to use them in the form of fusion proteins. In someembodiments, a lipocalin mutein of the disclosure is fused at itsN-terminus or its C-terminus to a protein, a protein domain or apeptide, for instance, a signal sequence and/or an affinity tag.

Affinity tags such as the Strep-tag or Strep-tag II (Schmidt et al., JMol Biol, 1996), the c-myc-tag, the FLAG-tag, the His-tag or the HA-tagor proteins such as glutathione-S-transferase, which allow easydetection and/or purification of recombinant proteins, are furtherexamples of suitable fusion partners. Finally, proteins with chromogenicor fluorescent properties such as the green fluorescent protein (GFP) orthe yellow fluorescent protein (YFP) are suitable fusion partners forlipocalin muteins of the disclosure as well.

In general, it is possible to label the lipocalin muteins of thedisclosure with any appropriate chemical substance or enzyme, whichdirectly or indirectly generates a detectable compound or signal in achemical, physical, optical, or enzymatic reaction. An example for aphysical reaction and at the same time optical reaction/marker is theemission of fluorescence upon irradiation or the emission of x-rays whenusing a radioactive label. Alkaline phosphatase, horseradish peroxidaseand p-g a lactosid ase are examples of enzyme labels (and at the sametime optical labels) which catalyze the formation of chromogenicreaction products. In general, all labels commonly used for antibodies(except those exclusively used with the sugar moiety in the Fc part ofimmunoglobulins) can also be used for conjugation to the lipocalinmuteins of the disclosure. The lipocalin muteins of the disclosure mayalso be conjugated with any suitable therapeutically active agent, e.g.,for the targeted delivery of such agents to a given cell, tissue ororgan, or for the selective targeting of cells (e.g., tumor cells)without affecting the surrounding normal cells. Examples of suchtherapeutically active agents include radionuclides, toxins, smallorganic molecules, and therapeutic peptides (such as peptides acting asagonists/antagonists of a cell surface receptor or peptides competingfor a protein binding site on a given cellular target). The lipocalinmuteins of the disclosure may, however, also be conjugated withtherapeutically active nucleic acids such as antisense nucleic acidmolecules, small interfering RNAs, micro RNAs or ribozymes. Suchconjugates can be produced by methods well known in the art.

As indicated above, a lipocalin mutein of the disclosure may in someembodiments be conjugated to a moiety that extends the serum half-lifeof the mutein (in this regard see also International Patent PublicationNo. WO 2006/056464, where such conjugation strategies are described withreference to muteins of human neutrophil gelatinase-associated lipocalin(hNGAL) with binding affinity for CTLA-4). The moiety that extends theserum half-life may be a polyalkylene glycol molecule, hydroxyethylstarch, fatty acid molecules, such as palmitic acid (Vajo and Duckworth,Pharmacol Rev, 2000), an Fc part of an immunoglobulin, a C_(H)3 domainof an immunoglobulin, a C_(H)4 domain of an immunoglobulin, an albuminbinding peptide, or an albumin binding protein, transferrin to name onlya few. The albumin binding protein may be a bacterial albumin bindingprotein, an antibody, an antibody fragment including domain antibodies(e.g., U.S. Pat. No. 6,696,245), or a lipocalin mutein with bindingactivity for albumin. Accordingly, suitable conjugation partners forextending the half-life of a lipocalin mutein of the disclosure includean albumin binding protein, for example, a bacterial albumin bindingdomain, such as the one of streptococcal protein G (Konig and Skerra, JImmunol Methods, 1998). Other examples of albumin binding peptides thatcan be used as conjugation partner are, for instance, those having aCys-Xaa₁-Xaa₂-Xaa₃-Xaa₄-Cys consensus sequence, wherein Xaa₁ is Asp,Asn, Ser, Thr, or Trp; Xaa₂ is Asn, Gln, His, Ile, Leu, or Lys; Xaa₃ isAla, Asp, Phe, Trp, or Tyr; and Xaa₄ is Asp, Gly, Leu, Phe, Ser, or Thras described in U.S. Patent Publication No. 20030069395 or Dennis et al.(J Biol Chem, 2002).

In other embodiments, albumin itself (Osborn et al., J Pharmacol ExpTher, 2002), or a biologically active fragment of albumin can be used asconjugation partner of a lipocalin mutein of the disclosure. The term“albumin” includes all mammal albumins such as human serum albumin orbovine serum albumin or rat albumin. The albumin or fragment thereof canbe recombinantly produced as described in U.S. Pat. No. 5,728,553 orEuropean Patent Publication Nos. EP0330451 and EP0361991. Recombinanthuman albumin (e.g., Recombumin® from Novozymes Delta Ltd., Nottingham,UK) can be conjugated or fused to a lipocalin mutein of the disclosurein order to extend the half-life of the mutein.

If the albumin-binding protein is an antibody fragment it may be adomain antibody. Domain Antibodies (dAbs) are engineered to allowprecise control over biophysical properties and in vivo half-life tocreate the optimal safety and efficacy product profile. DomainAntibodies are for example commercially available from Domantis Ltd.(Cambridge, UK and Mass., USA).

If a transferrin is used as a moiety to extend the serum half-life ofthe lipocalin muteins of the disclosure, the muteins can be geneticallyfused to the N- or C-terminus, or both, of non-glycosylated transferrin.Non-glycosylated transferrin has a half-life of 14-17 days, and atransferrin fusion protein will similarly have an extended half-life.The transferrin carrier also provides high bioavailability,biodistribution, and circulating stability. This technology iscommercially available from BioRexis (BioRexis PharmaceuticalCorporation, PA, USA). Recombinant human transferrin (DeltaFerrin™) foruse as a protein stabilizer/half-life extension partner is alsocommercially available from Novozymes Delta Ltd. (Nottingham, UK).

If an Fc part of an immunoglobulin is used for the purpose to prolongthe serum half-life of the lipocalin muteins of the disclosure, theSynFusion™ technology, commercially available from SyntonixPharmaceuticals, Inc. (MA, USA), may be used. The use of this Fc-fusiontechnology allows the creation of longer-acting biopharmaceuticals andmay for example consist of two copies of the mutein linked to the Fcregion of an antibody to improve pharmacokinetics, solubility, andproduction efficiency.

Yet another alternative to prolong the half-life of the lipocalinmuteins of the disclosure is to fuse to the N- or C-terminus of a muteina long, unstructured, flexible glycine-rich sequences (for examplepoly-glycine with about 20 to 80 consecutive glycine residues). Thisapproach disclosed in International Patent Publication No. WO2007/038619, for example, has also been term “rPEG” (recombinant PEG).

If PEG is used as conjugation partner, the polyalkylene glycol can besubstituted, unsubstituted, linear, or branched. It can also be anactivated polyalkylene derivative. Examples of suitable compounds arePEG molecules as described in International Patent Publication No. WO99/64016, in U.S. Pat. No. 6,177,074, or in U.S. Pat. No. 6,403,564 inrelation to interferon, or as described for other proteins such asPEG-modified asparaginase, PEG-adenosine deaminase (PEG-ADA) orPEG-superoxide dismutase (Fuertges and Abuchowski, Journal of ControlledRelease, 1990). The molecular weight of such a polymer, such as PEG, mayrange from about 300 to about 70,000 daltons, including, for example,polyethylene glycol with a molecular weight of about 10,000, of about20,000, of about 30,000 or of about 40,000 daltons. Moreover, as e.g.,described in U.S. Pat. Nos. 6,500,930 or 6,620,413, carbohydrateoligomers and polymers such as HES can be conjugated to a mutein of thedisclosure for the purpose of serum half-life extension.

In addition, a lipocalin mutein disclosed herein may be fused to amoiety may confer new characteristics to the lipocalin muteins of thedisclosure such as enzymatic activity or binding affinity for othertargets. Examples of suitable fusion partners are alkaline phosphatase,horseradish peroxidase, glutathione S-transferase, the albumin-bindingdomain of protein G, protein A, antibodies or antibody fragments,oligomerization domains, or toxins.

In particular, it may be possible to fuse a lipocalin mutein disclosedherein with a separate enzyme active site such that both “components” ofthe resulting fusion protein together act on a given therapeutic target.The binding domain of the lipocalin mutein attaches to thedisease-causing target, allowing the enzyme domain to abolish thebiological function of the target.

The present disclosure also relates to nucleic acid molecules (DNA andRNA) that include nucleotide sequences encoding the lipocalin muteins ofthe disclosure. Since the degeneracy of the genetic code permitssubstitutions of certain codons by other codons specifying the sameamino acid, the disclosure is not limited to a specific nucleic acidmolecule encoding a lipocalin mutein as described herein but encompassesall nucleic acid molecules that include nucleotide sequences encoding afunctional mutein. In this regard, the present disclosure providesnucleotide sequences encoding some lipocalin muteins of the disclosureas shown in SEQ ID NOs: 29-50, 71-84, and 96-106.

In another embodiment of the method according to the disclosure, anucleic acid molecule encoding a hTlc is firstly subjected tomutagenesis at one or more of the amino acid sequence positions 5, 7-8,10, 14, 16, 25-34, 44, 46, 52-53, 55-56, 58, 60-61, 63, 65-66, 69-70,73, 79-80, 84-86, 89-90, 93, 96-98, 101, 105-106, 108, 110-114, 121,124, 148-150, 152-154, and 157 of the linear polypeptide sequence ofmature hTlc (SEQ ID NO: 1). Secondly, the nucleic acid molecule encodinga human tear lipocalin is also subjected to mutagenesis at one or moreof the amino acid sequence positions 101, 111, 114 and 153 of the linearpolypeptide sequence of mature hTlc (SEQ ID NO:1).

The disclosure also includes nucleic acid molecules encoding thelipocalin muteins of the disclosure, which include additional mutationsoutside the indicated sequence positions of experimental mutagenesis.Such mutations are often tolerated or can even prove to be advantageous,for example if they contribute to an improved folding efficiency, serumstability, thermal stability, formulation stability or ligand bindingaffinity of the muteins.

A nucleic acid molecule disclosed in this application may be “operablylinked” to one or more regulatory sequence(s) to allow expression ofthis nucleic acid molecule.

A nucleic acid molecule, such as DNA, is referred to as “capable ofexpressing a nucleic acid molecule” or “able to allow expression of anucleotide sequence” if it includes sequence elements that containinformation regarding to transcriptional and/or translationalregulation, and such sequences are “operably linked” to the nucleotidesequence encoding the polypeptide. An operable linkage is a linkage inwhich the regulatory sequence elements and the sequence to be expressedare connected in a way that enables gene expression. The precise natureof the regulatory regions necessary for gene expression may vary amongspecies, but in general these regions include a promoter, which, inprokaryotes, contains both the promoter per se, i.e., DNA elementsdirecting the initiation of transcription, as well as DNA elementswhich, when transcribed into RNA, will signal the initiation oftranslation. Such promoter regions normally include 5′ non-codingsequences involved in initiation of transcription and translation, suchas the -35/-10 boxes and the Shine-Dalgarno element in prokaryotes orthe TATA box, CAAT sequences, and 5′ capping elements in eukaryotes.These regions can also include enhancer or repressor elements as well astranslated signal and leader sequences for targeting the nativepolypeptide to a specific compartment of a host cell.

In addition, the 3′ non-coding sequences may contain regulatory elementsinvolved in transcriptional termination, polyadenylation or the like.If, however, these termination sequences are not satisfactory functionalin a particular host cell, then they may be substituted with signalsfunctional in that cell.

Therefore, a nucleic acid molecule of the disclosure can include aregulatory sequence, such as a promoter sequence. In some embodiments, anucleic acid molecule of the disclosure includes a promoter sequence anda transcriptional termination sequence. Suitable prokaryotic promotersare, for example, the tet promoter, the IacUV5 promoter, or the T7promoter. Examples of promoters useful for expression in eukaryoticcells are the SV40 promoter or the CMV promoter.

The nucleic acid molecules of the disclosure can also be part of avector or any other kind of cloning vehicle, such as a plasmid, aphagemid, a phage, a baculovirus, a cosmid, or an artificial chromosome.

In one embodiment, the nucleic acid molecule is included in a phasmid. Aphasmid vector denotes a vector encoding the intergenic region of atemperent phage, such as M13 or f1, or a functional part thereof fusedto the cDNA of interest. After superinfection of the bacterial hostcells with such an phagemid vector and an appropriate helper phage(e.g., M13K07, VCS-M13 or R408) intact phage particles are produced,thereby enabling physical coupling of the encoded heterologous cDNA toits corresponding polypeptide displayed on the phage surface (Lowman,Annu Rev Biophys Biomol Struct, 1997, Rodi and Makowski, Curr OpinBiotechnol, 1999).

Such cloning vehicles can include, aside from the regulatory sequencesdescribed above and a nucleic acid sequence encoding a lipocalin muteinas described herein, replication and control sequences derived from aspecies compatible with the host cell that is used for expression aswell as selection markers conferring a selectable phenotype ontransformed or transfected cells. Large numbers of suitable cloningvectors are known in the art, and are commercially available.

The DNA molecule encoding a lipocalin mutein as described herein, and inparticular a cloning vector containing the coding sequence of such amutein can be transformed into a host cell capable of expressing thegene. Transformation can be performed using standard techniques. Thus,the disclosure is also directed to a host cell containing a nucleic acidmolecule as disclosed herein.

The transformed host cells are cultured under conditions suitable forexpression of the nucleotide sequence encoding a fusion protein of thedisclosure. Suitable host cells can be prokaryotic, such as Escherichiacoli (E. coli) or Bacillus subtilis, or eukaryotic, such asSaccharomyces cerevisiae, Pichia pastoris, SF9 or High5 insect cells,immortalized mammalian cell lines (e.g., HeLa cells or CHO cells) orprimary mammalian cells.

The disclosure also relates to a method for the production of alipocalin mutein as described herein, wherein the mutein, a fragment ofthe mutein or a fusion protein of the mutein and another polypeptide(e.g., another lipocalin mutein or antibody or antibody fragment) isproduced starting from the nucleic acid coding for the mutein by meansof genetic engineering methods. The method can be carried out in vivo,the lipocalin mutein can for example be produced in a bacterial oreukaryotic host organism and then isolated from this host organism orits culture. It is also possible to produce a protein in vitro, forexample by use of an in vitro translation system.

When producing the lipocalin mutein in vivo a nucleic acid encoding suchmutein is introduced into a suitable bacterial or eukaryotic hostorganism by means of recombinant DNA technology (as already outlinedabove). For this purpose, the host cell is first transformed with acloning vector that includes a nucleic acid molecule encoding alipocalin mutein as described herein using established standard methods.The host cell is then cultured under conditions, which allow expressionof the heterologous DNA and thus the synthesis of the correspondingpolypeptide. Subsequently, the polypeptide is recovered either from thecell or from the cultivation medium.

In some embodiments, a nucleic acid molecule, such as DNA, disclosed inthis application may be “operably linked” to another nucleic acidmolecule of the disclosure to allow expression of a fusion protein ofthe disclosure. In this regard, an operable linkage is a linkage inwhich the sequence elements of the first nucleic acid molecule and thesequence elements of the second nucleic acid molecule are connected in away that enables expression of the fusion protein as a singlepolypeptide.

In addition, in some embodiments for hTlc muteins of the disclosure, thenaturally occurring disulfide bond between Cys 61 and Cys 153 may beremoved. Accordingly, such muteins can be produced in a cell compartmenthaving a reducing redox milieu, for example, in the cytoplasm ofGram-negative bacteria.

In case a lipocalin mutein of the disclosure includes intramoleculardisulfide bonds, it may be preferred to direct the nascent polypeptideto a cell compartment having an oxidizing redox milieu using anappropriate signal sequence. Such an oxidizing environment may beprovided by the periplasm of Gram-negative bacteria such as E. coli, inthe extracellular milieu of Gram-positive bacteria or in the lumen ofthe endoplasmic reticulum of eukaryotic cells and usually favors theformation of structural disulfide bonds.

It is, however, also possible to produce a mutein of the disclosure inthe cytosol of a host cell, preferably E. coli. In this case, thepolypeptide can either be directly obtained in a soluble and foldedstate or recovered in form of inclusion bodies, followed by renaturationin vitro. A further option is the use of specific host strains having anoxidizing intracellular milieu, which may thus allow the formation ofdisulfide bonds in the cytosol (Venturi et al., J Mol Biol, 2002).

However, a lipocalin mutein as described herein may not necessarily begenerated or produced only by use of genetic engineering. Rather, such amutein can also be obtained by chemical synthesis such as Merrifieldsolid phase polypeptide synthesis or by in vitro transcription andtranslation. It is for example possible that promising mutations areidentified using molecular modeling, polypeptides continuing suchmutations synthesized in vitro, and investigated for binding activitywith respect to LAG-3 and other desirable properties (such asstability). Methods for the solid phase and/or solution phase synthesisof polypeptides/proteins are well known in the art (see e.g.,Bruckdorfer et al., Curr Pharm Biotechnol, 2004).

In another embodiment, the lipocalin muteins of the disclosure may beproduced by in vitro transcription/translation employingwell-established methods known to those skilled in the art.

The skilled worker will appreciate methods useful to prepare lipocalinmuteins contemplated by the present disclosure but whose protein ornucleic acid sequences are not explicitly disclosed herein. As anoverview, such modifications of the amino acid sequence include, e.g.,directed mutagenesis of single amino acid positions in order to simplifysub-cloning of a mutated lipocalin gene or its parts by incorporatingcleavage sites for certain restriction enzymes. In addition, thesemutations can also be incorporated to further improve the affinity of alipocalin mutein for its target (e.g., LAG-3). Furthermore, mutationscan be introduced to modulate certain characteristics of the mutein suchas to improve folding stability, serum stability, protein resistance orwater solubility or to reduce aggregation tendency, if necessary. Forexample, naturally occurring cysteine residues may be mutated to otheramino acids to prevent disulphide bridge formation.

The lipocalin muteins disclosed herein and its derivatives can be usedin many fields similar to antibodies or fragments thereof. For example,the lipocalin muteins can be used for labeling with an enzyme, anantibody, a radioactive substance or any other group having biochemicalactivity or defined binding characteristics. By doing so, theirrespective targets or conjugates or fusion proteins thereof can bedetected or brought in contact with them. In addition, lipocalin muteinsof the disclosure can serve to detect chemical structures by means ofestablished analytical methods (e.g., ELISA or Western Blot) or bymicroscopy or immunosensorics. In this regard, the detection signal caneither be generated directly by use of a suitable mutein conjugate orfusion protein or indirectly by immunochemical detection of the boundmutein via an antibody.

Additional objects, advantages, and features of this disclosure willbecome apparent to those skilled in the art upon examination of thefollowing Examples and the attached Figures thereof, which are notintended to be limiting. Thus, it should be understood that although thepresent disclosure is specifically disclosed by exemplary embodimentsand optional features, modification and variation of the disclosuresembodied therein herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this disclosure.

The present invention may further be characterized by the followingitems:

Item 1. A lipocalin mutein that is capable of binding LAG-3 with anaffinity measured by K_(d) of about 250 nM or lower.

Item 2. The lipocalin mutein of item 1, wherein the mutein is capable ofbinding LAG-3 with an affinity measured by K_(d) of about 50 nM orlower.

Item 3. The lipocalin mutein of item 1, wherein the mutein is capable ofbinding LAG-3 with an affinity measured by K_(d) of about 3 nM or lower.

Item 4. The lipocalin mutein of item 1, wherein the mutein is capable ofbinding LAG-3 with an affinity measured by K_(d) of about 0.1 nM orlower.

Item 5. The lipocalin mutein of item 1, wherein the mutein is capable ofbinding LAG-3 with an affinity measured by K_(d) of about 0.05 nM orlower.

Item 6. The lipocalin mutein of any one of items 1-5, wherein the K_(d)values are determined by surface plasmon resonance analysis asessentially described in Example 4.

Item 7. The lipocalin mutein of any one of items 1-7, wherein the muteincomprises at least two or more mutated amino acid residues at thesequence positions 5, 7-8, 10, 14, 16, 25-34, 44, 46, 52-53, 55-56, 58,60-61, 63, 65-66, 69-70, 73, 79-80, 84-86, 89-90, 93, 96-98, 101,105-106, 108, 110-114, 121, 124, 148-150, 152-154, and 156-157 of thelinear polypeptide sequence of human tear lipocalin (SEQ ID NO: 1).

Item 8. The lipocalin mutein of any one of items 1-6, wherein the muteincomprises at least one mutated amino acid residues at the sequencepositions 14, 25-26, 28, 31-32, 52, 55, 58, 66, 79, 84, 86, 101,105-106, 108, 110, 112-114, and 121 of the linear polypeptide sequenceof human tear lipocalin (SEQ ID NO: 1).

Item 9. The lipocalin mutein of any one of items 1-7, wherein the muteinfurther comprises at least one or more mutated amino acid residues atthe sequence positions 5, 7-8, 10, 16, 26-34, 44, 46, 53, 56, 58, 60-61,63, 65-66, 69-70, 73, 79-80, 85, 89-90, 93, 96-98, 101, 105-106, 108,110-111, 114, 121, 124, 148-150, 152-154, and 156-157 of the linearpolypeptide sequence of human tear lipocalin (SEQ ID NO: 1).

Item 10. The lipocalin mutein of any one of items 1-9, wherein themutein comprises at least at least one or more mutated amino acidresidues at the sequence positions 5, 7-8, 10, 16, 44, 46, 63, 65,69-70, 73, 80, 84, 89-90, 93, 96-98, 113, 124, 148-150, 152, 154, and156-157 of the linear polypeptide sequence of hTlc (SEQ ID NO: 1).

Item 11. The lipocalin mutein of item 7, wherein the amino acid sequenceof the mutein comprises two or more of the following mutated amino acidresidues in comparison with the linear polypeptide sequence of humantear lipocalin (SEQ ID NO: 1): Ala 5→Thr; Asp 7→Gly; Glu 8→Gln; Ile10→Phe; Ser 14→Pro; Thr 16→Met; Asp 25→Ser; Arg 26→Ser, Asp, Glu, Ala,or Gly; Glu 27→Asp; Phe 28→Cys or Asp; Pro 29→Phe; Glu 30→Trp; Met31→Ile or Leu; Asn 32→Asp, Met or Thr; Leu 33→Asp; Glu 34→Val; Leu44→His; Gly 46→Asp; Lys 52→Arg; Val 53→Ala; Met 55→Val; Leu 56→Asp; Ser58→Phe or Asp; Arg 60→Phe; Cys 61→Trp; Glu 63→Asp; Lys 65→Glu; Ala66→Asn; Glu 69→Gly; Lys 70→Arg; Glu 73→Ala; Ala 79→Thr or Glu; Asp80→Gly; His 84→Tyr or Leu; Val 85→Ala or Asp; Ala 86→Asp; Ile 89→Ser orAsn; Arg 90→Ser; Val 93→Glu; His 96→Asn; Tyr 97→His; Ile 98→Val; Cys101→Ser or Phe; Leu 105→Cys or Gly; His 106→Ala, Gln, Glu, Lys, or Pro;Lys 108→Tyr or Thr; Val 110→Gly or Asn; Arg 111→Pro; Gly 112→Met, Val,or Leu; Val 113→Ala or Leu; Lys 114→Trp or Ala; Lys 121→Thr; Leu124→Gln; Arg 148→Trp; Gln 149→Leu; Ser 150→Gly; Thr 152→Pro; Cys153→Ser; Ser 154→Ala; insertion of Pro between positions 156→and 157.

Item 12. The lipocalin mutein of item 7, wherein the amino acid sequenceof the mutein comprises at least one of the following mutated amino acidresidues in comparison with the linear polypeptide sequence of humantear lipocalin (SEQ ID NO: 1): Ser 14→Pro; Asp 25→Ser; Arg 26→Ser, Asp,Glu, Ala, or Gly; Phe 28→Asp; Met 31→Leu; Asn 32→Met or Thr; Lys 52→Arg;Met 55→Val; Ser 58→Asp; Ala 66→Asn; Ala 79→Glu; His 84→Tyr or Leu; Ala86→Asp; Cys 101→Phe; Leu 105→Gly; His 106→Gln, Glu, Lys, or Pro; Lys108→Thr; Val 110→Gly or Asn; Gly 112→Met, Val, or Leu; Val 113→Ala orLeu; Lys 114→Ala; Lys 121→Thr.

Item 13. The lipocalin mutein of item 7, wherein the amino acid sequenceof the mutein comprises at least one of the following mutated amino acidresidues in comparison with the linear polypeptide sequence of humantear lipocalin (SEQ ID NO: 1): Ala 5 Thr; Asp 7→Gly; Glu 8→Gln; Ile10→Phe; Thr 16→Met; Arg 26→Ser; Glu 27→Asp; Phe 28→Cys; Pro 29→Phe; Glu30→Trp; Met 31→Ile; Asn 32→Asp; Leu 33→Asp; Glu 34→Val; Leu 44→His; Gly46→Asp; Val 53→Ala; Leu 56→Asp; Ser 58→Phe; Arg 60→Phe; Cys 61→Trp; Glu63→Asp; Lys 65→Glu; Glu 69→Gly; Lys 70→Arg; Glu 73→Ala; Ala 79→Thr; Asp80→Gly; Val 85→Ala or Asp; Ile 89→Ser or Asn; Arg 90→Ser; Val 93→Glu;His 96→Asn; Tyr 97→His; Ile 98→Val; Cys 101→Ser; Leu 105→Cys; His106→Ala; Lys 108→Tyr; Arg 111→Pro; Lys 114→Trp; Leu 124→Gln; Arg148→Trp; Gln 149→Leu; Ser 150→Gly; Thr 152→Pro; Cys 153→Ser; Ser154→Ala; insertion of Pro between positions 156 and 157.

Item 14. The lipocalin mutein of any one of items 1-13, wherein thelipocalin mutein binds LAG-3 with an EC₅₀ value of about 320 nM orlower.

Item 15. The lipocalin mutein of any one of items 14, wherein thelipocalin mutein binds LAG-3 with an EC₅₀ value of about 10 nM or lower.

Item 16. The lipocalin mutein of any one of items 14, wherein thelipocalin mutein binds LAG-3 with an EC₅₀ value of about 0.2 nM orlower.

Item 17. The lipocalin mutein of any one of items 14-16, wherein thesaid EC₅₀ values are measured by fluorescence-activated cell sorting asessentially described in Example 5.

Item 18. The lipocalin mutein of any one of items 1-17, wherein themutein is cross-reactive with both human LAG-3 and cynomolgus LAG-3 (SEQID NO: 1).

Item 19. The lipocalin mutein of any one of items 1-18, wherein themutein is capable of interfering with the binding of human LAG-3 tomajor histocompatibility complex (MHC) class II.

Item 20. The lipocalin mutein of item 19, wherein the capability ofinterfering with the binding of human LAG-3 to major histocompatibilitycomplex (MHC) class II is analyzed by fluorescence-activated cellsorting as essentially described in Example 6.

Item 21. The lipocalin mutein of any one of items 1-20, wherein theamino acid sequence of the mutein comprises the following amino acidmutations: Arg 26→Ser; Glu 27→Asp; Phe 28→Cys; Pro 29→Phe; Glu 30→Trp;Met 31→Ile; Asn 32→Asp; Leu 33→Asp; Glu 34→Val; Leu 56→Asp; Ser 58→Phe;Arg 60→Phe; Cys 61→Trp; Cys 101→Ser; Leu 105→Cys; His 106→Ala; Lys108→Tyr; Arg 111→Pro; Lys 114→Trp; Cys 153→Ser; and one or more of thefollowing amino acid mutations: Ala 5→Thr; Asp 7→Gly; Glu 8→Gln; Ile10→Phe; Thr 16→Met; Leu 44→His; Gly 46→Asp; Val 53→Ala; Glu 63→Asp; Lys65→Glu; Glu 69→Gly; Lys 70→Arg; Glu 73→Ala; Ala 79→Thr; Asp 80→Gly; Val85→Ala or Asp; Ile 89→Ser or Asn; Arg 90→Ser; Val 93→Glu; His 96→Asn;Tyr 97→His; Ile 98→Val; Leu 124→Gln; Arg 148→Trp; Gln 149→Leu; Ser150→Gly; Thr 152→Pro; Ser 154→Ala; insertion of Pro between positions156 and 157.

Item 22. The lipocalin mutein of any one of items 1-20, wherein theamino acid sequence of the mutein comprises the following amino acidmutations: Ser 14→Pro; Asp 25→Ser; Phe 28→Asp; Lys 52→Arg; Met 55→Val;Ser 58→Asp; Ala 66→Asn; Ala 79→Glu; Ala 86→Asp; Cys 101→Phe; Leu105→Gly; Lys 108→Thr; Lys 114→Ala; Lys 121→Thr; and one or more of thefollowing amino acid mutations: Arg 26→Ser, Asp, Glu, or Ala; Met31→Leu; Asn 32→Thr; Leu 56→Asp; His 84→Tyr or Leu; His 106→Glu, Lys, orPro; Val 110→Asn; Gly 112→Val or Leu; Val 113→Ala or Leu.

Item 23. The lipocalin mutein of any one of items 1-22, wherein theamino acid sequence of the mutein comprises one of the following sets ofamino acid mutations:

-   -   (a) Ala 5→Thr; Glu 8→Gln; Arg 26→Ser; Glu 27→Asp; Phe 28→Cys;        Pro 29→Phe; Glu 30→Trp; Met 31→Ile; Asn 32→Asp; Leu 33→Asp; Glu        34→Val; Leu 56→Asp; Ser 58→Phe; Arg 60→Phe; Cys 61→Trp; Lys        65→Glu; Glu 69→Gly; Val 85→Ala; Cys 101→Ser; Leu 105→Cys; His        106→Ala; Lys 108→Tyr; Arg 111→Pro; Lys 114→Trp; Cys 153→Ser; Ser        154→Ala; insertion of Pro between positions 156 and 157;    -   (b) Ala 5→Thr; Arg 26→Ser; Glu 27→Asp; Phe 28→Cys; Pro 29→Phe;        Glu 30→Trp; Met 31→Ile; Asn 32→Asp; Leu 33→Asp; Glu 34→Val; Gly        46→Asp; Leu 56→Asp; Ser 58→Phe; Arg 60→Phe; Cys 61→Trp; Lys        65→Glu; Val 85→Ala; Cys 101→Ser; Leu 105→Cys; His 106→Ala; Lys        108→Tyr; Arg 111→Pro; Lys 114→Trp; Ser 150→Gly; Cys 153→Ser;        insertion of Pro between positions 156 and 157;    -   (c) Asp 7→Gly; Arg 26→Ser; Glu 27→Asp; Phe 28→Cys; Pro 29→Phe;        Glu 30→Trp; Met 31→Ile; Asn 32→Asp; Leu 33→Asp; Glu 34→Val; Leu        56→Asp; Ser 58→Phe; Arg 60→Phe; Cys 61→Trp; Val 85→Asp; Cys        101→Ser; Leu 105→Cys; His 106→Ala; Lys 108→Tyr; Arg 111→Pro; Lys        114→Trp; Arg 148→Trp; Thr 152→Pro; Cys 153→Ser; insertion of Pro        between positions 156 and 157;    -   (d) Ala 5→Thr; Arg 26→Ser; Glu 27→Asp; Phe 28→Cys; Pro 29→Phe;        Glu 30→Trp; Met 31→Ile; Asn 32→Asp; Leu 33→Asp; Glu 34→Val; Val        53→Ala; Leu 56→Asp; Ser 58→Phe; Arg 60→Phe; Cys 61→Trp; Lys        65→Glu; Ala 79→Thr; Tyr 97→His; Cys 101→Ser; Leu 105→Cys; His        106→Ala; Lys 108→Tyr; Arg 111→Pro; Lys 114→Trp; Cys 153→Ser;        insertion of Pro between positions 156 and 157;    -   (e) Arg 26→Ser; Glu 27→Asp; Phe 28→Cys; Pro 29→Phe; Glu 30→Trp;        Met 31→Ile; Asn 32→Asp; Leu 33→Asp; Glu 34→Val; Leu 56→Asp; Ser        58→Phe; Arg 60→Phe; Cys 61→Trp; Glu 63→Asp; Val 85→Asp; Arg        90→Ser; His 96→Asn; Cys 101→Ser; Leu 105→Cys; His 106→Ala; Lys        108→Tyr; Arg 111→Pro; Lys 114→Trp; Leu 124→Gln; Cys 153→Ser;    -   (f) Thr 16→Met; Arg 26→Ser; Glu 27→Asp; Phe 28→Cys; Pro 29→Phe;        Glu 30→Trp; Met 31→Ile; Asn 32→Asp; Leu 33→Asp; Glu 34→Val; Leu        44→His; Leu 56→Asp; Ser 58→Phe; Arg 60→Phe; Cys 61→Trp; Lys        65→Glu; Ile 89→Ser; Cys 101→Ser; Leu 105→Cys; His 106→Ala; Lys        108→Tyr; Arg 111→Pro; Lys 114→Trp; Cys 153→Ser;    -   (g) Arg 26→Ser; Glu 27→Asp; Phe 28→Cys; Pro 29→Phe; Glu 30→Trp;        Met 31→Ile; Asn 32→Asp; Leu 33→Asp; Glu 34→Val; Leu 56→Asp; Ser        58→Phe; Arg 60→Phe; Cys 61→Trp; Glu 63→Asp; Lys 65→Glu; Cys        101→Ser; Leu 105→Cys; His 106→Ala; Lys 108→Tyr; Arg 111→Pro; Lys        114→Trp; Gln 149→Leu; Cys 153→Ser; insertion of Pro between        positions 156 and 157;    -   (h) Arg 26→Ser; Glu 27→Asp; Phe 28→Cys; Pro 29→Phe; Glu 30→Trp;        Met 31→Ile; Asn 32→Asp; Leu 33→Asp; Glu 34→Val; Leu 56→Asp; Ser        58→Phe; Arg 60→Phe; Cys 61→Trp; Lys 65→Glu; Lys 70→Arg; Cys        101→Ser; Leu 105→Cys; His 106→Ala; Lys 108→Tyr; Arg 111→Pro; Lys        114→Trp; Cys 153→Ser;    -   (i) Ala 5→Thr; Arg 26→Ser; Glu 27→Asp; Phe 28→Cys; Pro 29→Phe;        Glu 30→Trp; Met 31→Ile; Asn 32→Asp; Leu 33→Asp; Glu 34→Val; Leu        56→Asp; Ser 58→Phe; Arg 60→Phe; Cys 61→Trp; Lys 65→Glu; Asp        80→Gly; Ile 89→Asn; Ile 98→Val; Cys 101→Ser; Leu 105→Cys; His        106→Ala; Lys 108→Tyr; Arg 111→Pro; Lys 114→Trp; Cys 153→Ser;        insertion of Pro between positions 156 and 157;    -   (j) Ile 10→Phe; Arg 26→Ser; Glu 27→Asp; Phe 28→Cys; Pro 29→Phe;        Glu 30→Trp; Met 31→Ile; Asn 32→Asp; Leu 33→Asp; Glu 34→Val; Leu        56→Asp; Ser 58→Phe; Arg 60→Phe; Cys 61→Trp; Lys 65→Glu; Glu        73→Ala; Ile 89→Asn; Val 93→Glu; Cys 101→Ser; Leu 105→Cys; His        106→Ala; Lys 108→Tyr; Arg 111→Pro; Lys 114→Trp; Cys 153→Ser;    -   (k) Ala 5→Thr; Glu 8→Gln; Arg 26→Ser; Glu 27→Asp; Phe 28→Cys;        Pro 29→Phe; Glu 30→Trp; Met 31→Ile; Asn 32→Asp; Leu 33→Asp; Glu        34→Val; Leu 56→Asp; Ser 58→Phe; Arg 60→Phe; Cys 61→Trp; Lys        65→Glu; Glu 69→Gly; Val 85→Ala; Cys 101→Ser; Leu 105→Cys; His        106→Ala; Lys 108→Tyr; Arg 111→Pro; Lys 114→Trp; Cys 153→Ser; Ser        154→Ala; insertion of Pro between positions 156 and 157;    -   (l) Arg 26→Ser; Glu 27→Asp; Phe 28→Cys; Pro 29→Phe; Glu 30→Trp;        Met 31→Ile; Asn 32→Asp; Leu 33→Asp; Glu 34→Val; Leu 56→Asp; Ser        58→Phe; Arg 60→Phe; Cys 61→Trp; Cys 101→Ser; Leu 105→Cys; His        106→Ala; Lys 108→Tyr; Arg 111→Pro; Lys 114→Trp; Cys 153→Ser;    -   (m) Ser 14→Pro; Asp 25→Ser; Arg 26→Asp; Phe 28→Asp; Asn 32→Thr;        Lys 52→Arg; Met 55→Val; Ser 58→Asp; Ala 66→Asn; Ala 79→Glu; His        84→Tyr; Ala 86→Asp; Cys 101→Phe; Leu 105→Gly; Lys 108→Thr; Val        110→Gly; Gly 112→Met; Lys 114→Ala; Lys 121→Thr;    -   (n) Ser 14→Pro; Asp 25→Ser; Arg 26→Glu; Phe 28→Asp; Met 31→Leu;        Asn 32→Thr; Lys 52→Arg; Met 55→Val; Ser 58→Asp; Ala 66→Asn; Ala        79→Glu; His 84→Tyr; Ala 86→Asp; Cys 101→Phe; Leu 105→Gly; His        106→Gln; Lys 108→Thr; Val 110→Gly; Gly 112→Met; Lys 114→Ala; Lys        121→Thr;    -   (o) Ser 14→Pro; Asp 25→Ser; Arg 26→Glu; Phe 28→Asp; Asn 32→Thr;        Lys 52→Arg; Met 55→Val; Ser 58→Asp; Ala 66→Asn; Ala 79→Glu; His        84→Tyr; Ala 86→Asp; Cys 101→Phe; Leu 105→Gly; His 106→Glu; Lys        108→Thr; Val 110→Gly; Gly 112→Val; Lys 114→Ala; Lys 121→Thr;    -   (p) Ser 14→Pro; Asp 25→Ser; Arg 26→Asp; Phe 28→Asp; Asn 32→Thr;        Lys 52→Arg; Met 55→Val; Ser 58→Asp; Ala 66→Asn; Ala 79→Glu; His        84→Tyr; Ala 86→Asp; Cys 101→Phe; Leu 105→Gly; His 106→Gln; Lys        108→Thr; Val 110→Gly; Gly 112→Leu; Lys 114→Ala; Lys 121→Thr;    -   (q) Ser 14→Pro; Asp 25→Ser; Arg 26→Ser; Phe 28→Asp; Asn 32→Thr;        Lys 52→Arg; Met 55→Val; Ser 58→Asp; Ala 66→Asn; Ala 79→Glu; His        84→Tyr; Ala 86→Asp; Cys 101→Phe; Leu 105→Gly; His 106→Gln; Lys        108→Thr; Val 110→Gly; Gly 112→Met; Lys 114→Ala; Lys 121→Thr;    -   (r) Ser 14→Pro; Asp 25→Ser; Arg 26→Ala; Phe 28→Asp; Asn 32→Thr;        Lys 52→Arg; Met 55→Val; Ser 58→Asp; Ala 66→Asn; Ala 79→Glu; His        84→Tyr; Ala 86→Asp; Cys 101→Phe; Leu 105→Gly; His 106→Lys; Lys        108→Thr; Val 110→Gly; Gly 112→Met; Lys 114→Ala; Lys 121→Thr;    -   (s) Ser 14→Pro; Asp 25→Ser; Phe 28→Asp; Asn 32→Thr; Lys 52→Arg;        Met 55→Val; Ser 58→Asp; Ala 66→Asn; Ala 79→Glu; Ala 86→Asp; Cys        101→Phe; Leu 105→Gly; His 106→Gln; Lys 108→Thr; Val 110→Asn; Gly        112→Met; Val 113→Ala; Lys 114→Ala; Lys 121→Thr;    -   (t) Ser 14→Pro; Asp 25→Ser; Arg 26→Gly; Phe 28→Asp; Met 31→Leu;        Asn 32→Thr; Lys 52→Arg; Met 55→Val; Ser 58→Asp; Ala 66→Asn; Ala        79→Glu; His 84→Tyr; Ala 86→Asp; Cys 101→Phe; Leu 105→Gly; His        106→Pro; Lys 108→Thr; Val 110→Gly; Gly 112→Met; Lys 114→Ala; Lys        121→Thr;    -   (u) Ser 14→Pro; Asp 25→Ser; Arg 26→Asp; Phe 28→Asp; Asn 32→Thr;        Lys 52→Arg; Met 55→Val; Ser 58→Asp; Ala 66→Asn; Ala 79→Glu; His        84→Leu; Ala 86→Asp; Cys 101→Phe; Leu 105→Gly; His 106→Gln; Lys        108→Thr; Val 110→Gly; Gly 112→Met; Val 113→Leu; Lys 114→Ala; Lys        121→Thr; or    -   (v) Ser 14→Pro; Asp 25→Ser; Arg 26→Gly; Phe 28→Asp; Asn 32→Met;        Lys 52→Arg; Met 55→Val; Ser 58→Asp; Ala 66→Asn; Ala 79→Glu; Ala        86→Asp; Cys 101→Phe; Leu 105→Gly; His 106→Gln; Lys 108→Thr; Val        110→Gly; Gly 112→Met; Lys 114→Ala; Lys 121→Thr.

Item 24. The lipocalin mutein of any one of items 1-22, wherein theamino acid sequence of the mutein comprises one of the following sets ofamino acid mutations:

-   -   (a) Ala 5→Thr; Glu 8→Gln; Lys 65→Glu; Glu 69→Gly; Val 85→Ala;        Ser 154→Ala; insertion of Pro between positions 156 and 157;    -   (b) Ala 5→Thr; Gly 46→Asp; Lys 65→Glu; Val 85→Ala; Ser 150→Gly;        insertion of Pro between positions 156 and 157;    -   (c) Asp 7→Gly; Val 85→Asp; Arg 148→Trp; Thr 152→Pro; insertion        of Pro between positions 156 and 157;    -   (d) Ala 5→Thr; Val 53→Ala; Lys 65→Glu; Ala 79→Thr; Tyr 97→His;        insertion of Pro between positions 156 and 157;    -   (e) Glu 63→Asp; Val 85→Asp; Arg 90→Ser; His 96→Asn; Leu 124→Gln;    -   (f) Thr 16→Met; Leu 44→His; Lys 65→Glu; Ile 89→Ser;    -   (g) Glu 63→Asp; Lys 65→Glu; Gln 149→Leu; insertion of Pro        between positions 156 and 157;    -   (h) Lys 65→Glu; Lys 70→Arg;    -   (i) Ala 5→Thr; Lys 65→Glu; Asp 80→Gly; Ile 89→Asn; Ile 98→Val;        insertion of Pro between positions 156 and 157;    -   (j) Ile 10→Phe; Lys 65→Glu; Glu 73→Ala; Ile 89→Asn; Val 93→Glu;    -   (k) Arg 26→Asp; Asn 32→Thr; His 84→Tyr; Val 110→Gly; Gly        112→Met;    -   (l) Arg 26→Glu; Met 31→Leu; Asn 32→Thr; His 84→Tyr; His 106→Gln;        Val 110→Gly; Gly 112→Met;    -   (m) Arg 26→Glu; Asn 32→Thr; His 84→Tyr; His 106→Glu; Gly        112→Val;    -   (n) Arg 26→Asp; Asn 32→Thr; His 84→Tyr; His 106→Gln; Val        110→Gly; Gly 112→Leu;    -   (o) Arg 26→Ser; Asn 32→Thr; His 84→Tyr; His 106→Gln; Val        110→Gly; Gly 112→Met;    -   (p) Arg 26→Ala; Asn 32→Thr; His 84→Tyr; His 106→Lys; Val        110→Gly; Gly 112→Met;    -   (q) Asn 32→Thr; His 106→Gln; Val 110→Asn; Gly 112→Met; Val        113→Ala;    -   (r) Arg 26→Gly; Met 31→Leu; Asn 32→Thr; His 84→Tyr; His 106→Pro;        Val 110→Gly; Gly 112→Met; or    -   (s) Arg 26→Asp; Asn 32→Thr; His 84→Leu; His 106→Gln; Val        110→Gly; Gly 112→Met; Val 113→Leu.

Item 25. The lipocalin mutein of any one of items 1-24, wherein themutein comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 8-18 and 20-28 or of a fragment or variantthereof.

Item 26. The lipocalin mutein according to any of items 1-25, whereinthe mutein has at least 85%, at least 90%, at least 95%, at least 97.5%or at least 99% sequence identity to an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: SEQ ID NOs: 8-18 and 20-28.

Item 27. The lipocalin mutein of any one of items 1-26, wherein themutein is conjugated to a compound selected from the group consisting ofan organic molecule, an enzyme label, a radioactive label, a coloredlabel, a fluorescent label, a chromogenic label, a luminescent label, ahapten, digoxigenin, biotin, a cytostatic agent, a toxin, a metalcomplex, a metal, and colloidal gold.

Item 28. The lipocalin mutein of any one of items 1-27, wherein themutein is fused at its N-terminus and/or its C-terminus to a fusionpartner that is a protein, a protein domain, or a peptide.

Item 29. The lipocalin mutein of any one of items 1-28, wherein themutein is fused at its N-terminus and/or its C-terminus to a fusionpartner that is an antibody or antibody fragment.

Item 30. The lipocalin mutein of any one of items 1-29, wherein themutein is conjugated to a compound that extends the serum half-life ofthe mutein.

Item 31. The lipocalin mutein of item 30, wherein the compound thatextends the serum half-life is selected from the group consisting of apolyalkylene glycol molecule, hydroethylstarch, a Fc part of animmunoglobulin, a C_(H)3 domain of an immunoglobulin, a C_(H)4 domain ofan immunoglobulin, an albumin binding peptide, and an albumin bindingprotein.

Item 32. The lipocalin mutein of item 31 wherein the polyalkylene glycolmolecule is polyethylene (PEG) or an activated derivative thereof.

Item 33. A nucleic acid molecule comprising a nucleotide sequenceencoding a lipocalin mutein of any one of items 1-32.

Item 34. An expression vector comprising the nucleic acid molecule ofitem 33.

Item 35. A host cell containing a nucleic acid molecule of item 34.

Item 36. A method of producing a lipocalin mutein according to any oneof items 1-32, wherein the mutein is produced starting from the nucleicacid coding for the mutein or fragment thereof by means of geneticengineering methods.

Item 37. A method of binding LAG-3 in a subject, comprising applying oneor more lipocalin muteins according to any one of items 1-32 or one ormore compositions comprising such muteins.

Item 38. A method of stimulating immune response in a subject,comprising applying one or more lipocalin muteins according to any oneof items 1-32 or one or more compositions comprising such muteins.

Item 39. A method of inducing T lymphocyte proliferation in a subject,comprising applying one or more lipocalin muteins according to any oneof items 1-32 or one or more compositions comprising such muteins.

Item 40. A method of interfering with the binding of human LAG-3 tomajor histocompatibility complex (MHC) class II in a subject, comprisingapplying one or more lipocalin muteins of any one of items 1-32 or oneor more compositions comprising such muteins.

Item 41. The lipocalin mutein of any one of items 1-32 wherein themutein competes with the binding of human LAG-3 to cells expressingmajor histocompatibility complex (MHC) class II.

Item 42. The lipocalin mutein of any one of items 1-32 wherein themutein competes with the binding of human LAG-3 to cells expressingmajor histocompatibility complex (MHC) class II, when measured influorescence-activated cell sorting analysis as essentially described inExample 6.

Item 43. A pharmaceutical composition comprising a the lipocalin muteinof any one of items 1-32 and a pharmaceutically acceptable excipient.

Item 44. An immunoconjugate or fusion protein comprising the lipocalinmuteins, or fragment thereof, of any one of items 1-32 linked to atherapeutic agent.

Item 45. The use of a mutein according to any one of items 1-32 for thebinding/detection of LAG-3, comprising:

-   -   (a) contacting the mutein with a test sample suspected to        contain LAG-3, thereby allowing the formation of a complex        between the mutein and LAG-3; and    -   (b) detecting the complex between the mutein and LAG-3 by a        suitable signal.

Item 46. A diagnostic or analytical kit comprising a mutein according toany one of items 1-32.

Item 47. A method of detecting the presence of LAG-3 in a biologicalsample, the method comprising contacting the sample with a mutein of anyone of items 1-32 under conditions that allow the formation of a complexof the mutein and LAG-3.

Item 48. The method of item 47, further comprising detecting the complexof the mutein and LAG-3.

Item 49. The method of item 47 or 48, wherein the biological sample isisolated from a human.

Item 50. The method of any one of items 47-49, wherein the samplecomprises body fluid.

V. EXAMPLES Example 1: Generation of Maturation Libraries and Selectionof Optimized Muteins Specifically Binding to LAG-3

For optimization of LAG-3-specific muteins, libraries were generatedbased on mutein SEQ ID NOs: 7 or 19 using either a biased randomizationof selected positions or error prone polymerase chain reaction (PCR)based methods. The biased design was made such that for each of theselected positions the amino acid encoded corresponds to the amino acidfound in the respective mother clone with a probability of 50-70%, whileit can be a different amino acid with a 50-30% probability. With N asthe number of targeted positions and B as bias, the most probable numberof exchanges per clone is N×(1-B).

The generated lipocalin muteins were cloned with high efficiency intophagemid vector essentially as described (Kim et al., J Am Chem Soc,2009). Phage display was employed to select for optimized muteins withimproved heat stability and binding affinity. The phagemid selection wasconducted with increased stringency compared to the initial muteinselections and involved preincubation steps at elevated temperature andlimiting target concentration amongst other things.

Example 2: Identification of Muteins Specifically Binding to LAG-3 UsingHigh-Throughput ELISA Screening

Individual colonies were used to inoculate 2× Yeast Extract Trypton(2×YT)/Amp medium and grown overnight (14-18 h) to stationary phase.Subsequently, 50 μL 2×YT/Amp were inoculated from the stationary phasecultures and incubated for 3 h at 37° C. and then shifted to 22° C.until an OD₅₉₅ of 0.6-0.8 was reached. Production of muteins was inducedby addition of 10 μL 2×YT/Amp supplemented with 1.2 Σg/mLanhydrotetracycline. Cultures were incubated at 22° C. until the nextday. After addition of 40 μL of 5% (w/v) BSA in PBS/T and incubation for1 h at 25° C., cultures were ready for use in screening assays.

Reverse screening formats were applied, where the muteins were capturedvia the Strep-tag on microtiter plates coated with anti-Strep-Tagantibody and biotinylated LAG-3-Fc was added and detected viaExtravidin-horseradish peroxidase (HRP) (Sigma).

To select for muteins with increased affinity and stability thescreening was performed with i) reduced antigen concentration, ii) usingreverse screening formats where the muteins were captured via theStrep-tag on microtiter plates coated with anti-Strep-Tag antibody anddifferent concentrations of the target was added and detected via eitherExtravidin-HRP (Sigma) and partially iii) incubation of the screeningsupernatant at 75° C. before addition to the target plate.

Clones were then sequenced based on the screening results, and muteinswere selected for further characterization.

Example 3: Expression of Muteins

Selected muteins with C-terminal sequence SAWSHPQFEK of SA linker andthe Strep-tag II peptide (WSHPQFEK) were expressed in E. coli in2XYT/Amp medium to purify the muteins after expression usingStrep-Tactin affinity chromatography and preparative size exclusionchromatography (SEC). After SEC purification, the fractions containingmonomeric protein are pooled and analyzed again using analytical SEC.The yield of the lipocalin muteins after Strep-Tactin affinitychromatography and preparative size exclusion chromatography (SEC) isshown in Table 1 as well as the monomer content of the lipocalin muteinsafter Strep-Tactin purification:

TABLE 1 Expression of muteins Monomer content (assessed by Yieldanalytical SEC) SEQ ID NO: [mg/L] [%] 85 4.03 100 86 8.33 100 87 10.79100 88 10.63 94 89 9.56 89.6 90 10.40 89.9 91 9.96 95 92 8.75 98.9 9310.26 92.6 94 10.15 91.3 20 7.53 95 21 8.40 96 22 7.86 95 23 7.28 89 247.14 92 25 7.38 92 26 8.74 93 27 8.84 97 28 8.04 97 57 0.38 100 61 0.96100 63 2.26 100 64 0.64 100 65 5.85 100 66 2.20 100 67 0.69 100 68 8.29100

Example 4: Affinity of Muteins Binding to Human and Cynomolgus LAG-3Determined by Surface Plasmon Resonance (SPR)

Surface plasmon resonance (SPR) was used to measure binding kinetics andaffinity of the representative lipocalin muteins disclosed herein.

The binding of exemplary lipocalin muteins to huLAG-3-Fc (R&D Systems)and cyLAG-3-Fc was determined by Surface Plasmon Resonance (SPR) using aBiacore T200 instrument (GE Healthcare). Recombinant LAG-3 fromcynomolgus monkeys (cyLAG-3-Fc) was produced by fusing the extracellulardomain of cynomolgus LAG-3 (cyLAG-3) to human IgG1 Fc fragment via aFactor Xa cleavage site and a (G₄5)₃ linker.

The anti-human IgG Fc antibody (GE Healthcare) was immobilized on a CM5sensor chip using standard amine chemistry: the carboxyl groups on thechip were activated using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide(EDC) and N-hydroxysuccinimide (NHS). Subsequently, anti-human IgG Fcantibody solution (GE Healthcare) at a concentration of 25 μg/mL in 10mM sodium acetate (pH) 5 was applied at a flow rate of 5 μL/min until animmobilization level of 9000-14000 resonance units (RU) was achieved.Residual non-reacted NHS-esters were blocked by passing a solution of 1Methanolamine across the surface. The reference channel was treated in ananalogous manner. Subsequently, huLAG-3-Fc at 0.25 μg/mL or cyLAG-3-Fcat 1.5 μg/mL in HBS-EP+ buffer was captured by the anti-human IgG-Fcantibody at the chip surface for 180 s at a flow rate of 10 μL/min.

For affinity determination, dilutions of each mutein were prepared inHBS-EP+ buffer and applied to the prepared chip surface. For SEQ ID NOs:19-28 concentrations of 100 nM down to 4 nM and in some cases down to0.8 nM were applied and for SEQ ID NOs: 7 and 85-94 concentrations of 6nM down to 0.5 nM were applied for affinity measurement to human LAG-3and 8 nM down to 0.5 nM for affinity measurement to cynomolgus LAG-3.The binding assay was carried out with a contact time of 180 s, adissociation time of 1500 or 600 s and a flow rate of 30 μL/min. Allmeasurements were performed at 25° C. Regeneration of the chip surfacewas achieved with injections of 3 M MgCl₂ for 60 s and 10 mM glycine-HCl(pH 1.7) for 180 s at a flow rate of 10 μL/min followed by an extra washwith running buffer (HBS-EP+ buffer) and a stabilization period of 120s. Lipocalin mutein SEQ ID NO: 3 was also tested as a negative control.Prior to the protein measurements, three startup cycles were performedfor conditioning purposes. Data were evaluated with Biacore T200Evaluation software (v2.0). Double referencing was used and the 1:1binding model was used to fit the raw data.

The values determined for k_(on), k_(off) and the resulting equilibriumdissociation constant (K_(d)) for SEQ ID NOs: 7 and 19, and theoptimized lipocalin muteins of SEQ ID NOs: 85-94, 20-28, 57, 61, and63-68 are summarized in Table 2. All optimized LAG-3 specific lipocalinmuteins bind human as well as cynomolgus LAG-3 with picomolar to lownanomolar affinity and affinities are up to 60-fold improved afteroptimization.

TABLE 2 Kinetic constants and affinities of LAG-3-specific muteinsdetermined by surface-plasmon-resonance (SPR). human LAG-3 cynomolgusLAG-3 k_(on) k_(off) K_(d) k_(on) k_(off) K_(d) SEQ ID NO: [M⁻¹ · s⁻¹][s⁻¹] [nM] [M⁻¹ · s⁻¹] [s⁻¹] [nM] 7 5.86E+06 1.89E−03 0.32 2.29E+061.81E−01 78.99 85 5.30E+06 2.80E−04 0.053 4.62E+06 6.21E−03 1.35 864.78E+06 3.70E−04 0.077 4.44E+06 1.11E−02 2.51 87 5.85E+06 5.08E−040.087 4.27E+06 1.77E−02 4.15 88 5.81E+06 2.88E−04 0.05 4.83E+06 5.55E−031.15 89 4.93E+06 4.67E−04 0.095 4.39E+06 1.88E−02 4.281 90 4.75E+066.18E−04 0.13 7.74E+06 5.82E−02 7.517 91 5.56E+06 6.62E−04 0.1197.78E+06 5.33E−02 6.856 92 5.54E+06 8.09E−04 0.146 4.63E+07 4.94E−0110.681 93 4.41E+06 4.63E−04 0.105 5.22E+06 1.98E−02 3.789 94 4.87E+067.00E−04 0.144 2.47E+07 2.12E−01 8.605 19 2.62E+05 7.18E−04 2.7412.24E+05 6.74E−04 3.012 20 1.88E+05 1.35E−04 0.722 1.63E+05 7.51E−050.461 21 1.57E+05 1.13E−04 0.718 1.42E+05 6.62E−05 0.467 22 2.34E+051.45E−04 0.619 1.80E+05 9.14E−05 0.507 23 1.58E+05 1.05E−04 0.6681.22E+05 7.16E−05 0.589 24 2.07E+05 1.40E−04 0.676 1.42E+05 1.17E−040.826 25 1.10E+05 1.42E−04 1.29 1.03E+05 1.19E−04 1.161 26 1.01E+051.38E−04 1.366 9.41E+04 1.36E−04 1.45 27 1.21E+05 1.74E−04 1.4391.22E+05 2.41E−04 1.97 28 4.63E+05 4.09E−04 0.883 2.36E+05 4.01E−04 1.757 2.21E+06 5.81E−05 0.026 1.98E+06 1.89E−03 0.954 61 1.61E+07 1.90E−030.118 6.25E+06 1.28E−02 2.04 63 6.59E+07 7.22E−03 0.11 1.38E+07 2.56E−021.85 64 3.34E+07 7.85E−03 0.235 1.13E+07 8.16E−02 7.24 65 2.58E+075.01E−03 0.195 1.46E+07 6.41E−02 4.39 66 7.05E+07 5.59E−03 0.07932.01E+07 2.42E−02 1.2 67 2.43E+07 5.16E−03 0.213 1.08E+07 5.53E−02 5.1368 3.25E+07 5.15E−03 0.158 2.01E+07 4.33E−02 2.15

Example 5: Fluorescence-Activated Cell Sorting (FACS) Analysis ofLipocalin Muteins Binding to Cells Expressing Human and Cynomolgus LAG-3

We employed fluorescence-activated cell sorting (FACS) studies in orderto assess the specific binding of lipocalin muteins SEQ ID NOs: 7,19-28, and 85-94 to Chinese hamster ovary (CHO) cells stably transfectedwith huLAG-3 (CHO-huLAG-3) or cyLAG-3 (CHO-cyLAG-3). SEQ ID NO: 3 wastested in parallel as negative control. The cell lines were generatedusing the Flp-In system (Invitrogen) according to the manufacturer'sinstructions. Mock-transfected Flp-In CHO cells served as the negativecontrol.

Transfected CHO cells were maintained in Ham's F12 medium (Invitrogen)supplemented with 10% Fetal Calf Serum (FCS, Biochrom) and 500 μg/mLHygromycin B (Roth). Cells were cultured in cell culture flasks understandard conditions according to manufacturer's instruction (37° C., 5%CO₂ atmosphere). In order to dissociate the adherent cells forsubculture or FACS experiments, Accutase (PAA) was employed according tothe manufacturer's instructions.

To perform the experiment, LAG-3-positive and negative Flp-In CHO cellswere incubated with lipocalin muteins, and bound mutein was labeledusing fluorescently labeled anti-hTlc antibodies, and then the signalwas detected using FACS analysis as described in this example.

5×10⁴ cells per well were pre-incubated for 1 h in ice-cold PBScontaining 5% fetal calf serum (PBS-FCS). Subsequently, a dilutionseries of lipocalin muteins, the negative control lipocalin mutein (SEQID NO: 3), and a benchmark anti-LAG-3 antibody (SEQ ID NOs: 5 and 6)typically ranging from 1 μM to 0.01 nM, was added to the cells, andincubated on ice for 1 h. Cells were washed twice in ice-cold PBS usingcentrifugation at 500×g and then incubated with a rabbit anti-lipocalinantibody labeled with the fluorescent dye Alexa 488 (Pieris) or a goatanti-human IgG antibody labeled with Alexa 488 (Invitrogen) for 30 minon ice. Cells were subsequently washed and analyzed using a intellicytIQue Flow cytometer (Intellicyt). Fluorescent data generated bylipocalin mutein binding to LAG-3 expressing cells were analyzed bygating for LAG-3 expressing CHO cells and using Forecyt® software andresulted geometric fluorescent mean were plotted and fitted usingGraphpad software. Data generated for SEQ ID NOs: 7, 19-28, and 85-94are shown in FIG. 2A, FIG. 2B, and Table 3. All optimized LAG-3 specificmuteins (SEQ ID NOs: 85-94 and 20-28) show clear binding to CHO cellsexpressing either huLAG-3 or cyLAG-3, with EC₅₀ comparable to thebenchmark antibody. The majority of the optimized muteins exhibits lowerEC₅₀ values compared to SEQ ID NOs: 7 and 19, indicating improvedbinding as compared to the parental lipocalin muteins. The differencesbetween binding affinities to human and cynomolgus LAG-3, has beensignificantly reduced for most of the optimized muteins, representing apreferred feature for potential pharmacokinetic or drug-safety studies.The negative control lipocalin mutein (SEQ ID NO: 3), which do not bindLAG-3, did not show any binding (not shown). No binding of the lipocalinmuteins was detected on mock-transfected Flp-In CHO cells (not shown).

TABLE 3 Binding of LAG-3 specific lipocalin muteins and the referencemolecule (benchmark anti-LAG-3 antibody, SEQ ID NOs: 5 and 6) to CHOcells transfected with huLAG-3 or cynomolgus LAG. EC₅₀ [nM] EC₅₀ [nM]CHO::human CHO::cynomolgus SEQ ID NO: LAG-3 LAG-3 7 1.33 319.3 85 0.229.8 86 1.52 21.16 87 0.95 21.05 88 0.18 9.3 89 0.85 41.73 90 0,02 103.491 0.61 74.27 92 0.76 111.3 93 0.69 42.68 94 0.53 117.4 19 4.09 26.2 201.97 29.84 21 2.52 31.59 22 2.23 33.06 23 3.04 33.61 24 2.35 34.93 252.55 38.93 26 2.23 44.97 27 2.15 31.37 28 2.19 21.04 5 and 6 0.5 46.8

Example 6: FACS Analysis of Competitive Binding of Lipocalin Muteins forHuman LAG-3 with MHC Class II Expressing Cells

To assess whether a given lipocalin mutein interferes with LAG-3 bindingto MHC class II on MHC class II-positive cells, a competition FACSexperiment was utilized. In this experiment, a constant concentration ofhuman LAG-3-Fc fusion (huLAG-3-Fc, R&D system) and a dilution series ofeach lipocalin mutein were incubated with the MHC class II positivehuman cell line A375, and cell-bound huLAG-3-Fc was detected using afluorescently labelled anti-IgG Fc antibody. In this assay, competitivelipocalin muteins interfering with the binding of huLAG-3 with itsligand MHC class II lead to a reduction of huLAG-3-Fc binding to the MHCclass II positive cell line A375.

The melanoma cell line A375 was maintained in DMEM medium (Invitrogen)supplemented with 10% Fetal Calf Serum (FCS, Biochrom). Cells werecultured in cell culture flasks under standard conditions according tomanufacturer's instruction (37° C., 5% CO₂ atmosphere). In order todissociate the adherent cells for subculture or FACS experiments,Accutase (PAA Laboratories GmbH) was employed according to themanufacturer's instructions.

For FACS assay, 5×10⁴ A375 cells per well were incubated for 1 h inPBS-FCS, followed by addition of 3 nM huLAG-3-Fc and varyingconcentrations of the LAG-3-specific lipocalin muteins, ranging from 1μM to 0.01 nM. Cells were washed twice in ice-cold PBS, re-suspended inPBS-FCS and incubated 30 min on ice with phycoerythrin labelledanti-human IgG Fc antibody (Jackson ImmunoResearch). Cells weresubsequently washed and analyzed using an Intellicyt IQue Flow cytometer(Intellicyt). Fluorescent data generated by huLAG-3-Fc binding to A375cells were analyzed using Forecyt software, and resulted geometricfluorescent mean were normalized to huLAG-3-Fc maximal binding. Percentof huLAG-3-Fc binding were plotted and fitted using Graphpad software.IC₅₀ values of SEQ ID NOs: 7, 19-28, and 85-94 are summarized in Table 4and selected competition binding curves are provided in FIG. 3. The datashow that all optimized lipocalin muteins compete with binding ofhuLAG-3 to its ligand MHC class II on human MHC class II expressingcells. The detection limit for such experiment was reached, thusimprovement in IC₅₀ of optimized lipocalin muteins compared to parentalmuteins, if any, were not observed. The negative control lipocalinmutein (SEQ ID NO: 3), which does not bind to LAG-3, did not show anycompetition.

TABLE 4 Lipocalin muteins compete with binding of huLAG-3 to its ligandMHC class II on MHC class II expressing cells. IC₅₀ SEQ ID NO: [nM] 70.22 85 0.38 86 0.78 87 0.32 88 1.4 89 0.58 90 0.25 91 0.39 92 0.23 930.29 94 0.41 19 1.2 20 1.7 21 0.68 22 1.11 23 0.63 24 1.01 25 0.3 260.54 27 0.39 28 0.55 5 and 6 0.28

Example 7: Thermal Stability Assessment of Lipocalin Muteins

To determine the melting temperatures (T_(m)s) of the lipocalin muteins,which is a general indicator for overall stability, the LAG-3 specificmuteins, at a protein concentration of 1 mg/mL in PBS (Gibco), werescanned (25-100° C.) at 1° C./min using a capillary nanoDSC instrument(CSC 6300, TA Instruments). The T_(m)s were calculated from thedisplayed thermogram using the integrated Nano Analyze software.

The resulting maximum melting temperatures as well as the onset ofmelting for exemplary lipocalin muteins (SEQ ID NOs: 7,19-28,85-94, and67) are listed in Table 5 below. Almost all lipocalin muteins haveT_(m)s in the range of 60 to 80° C., indicating good overall stabilitywith respect to each of these muteins.

TABLE 5 T_(m) and onset melting temperature as determined by nanoDSC ofLAG-3-specific lipocalin muteins T_(m) Onset melting SEQ ID NO: [° C.][° C.] 7 73 and 81 58 85 72 60 86 74 60 87 72 61 88 68 55 89 65 54 90 66and 72 59 91 80 66 92 80 69 93 69 and 73 58 94 67 55 19 58 and 67 42 2064 and 69 49 21 58 and 69 50 22 64 56 23 55 and 67 47 24 59 and 68 51 2560 and 68 50 26 59 and 70 50 27 60 and 70 49 28 63 and 70 51 67 88 74

Embodiments illustratively described herein may suitably be practiced inthe absence of any element or elements, limitation or limitations, notspecifically disclosed herein. Thus, for example, the terms“comprising,” “including,” “containing,” etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present embodiments have been specificallydisclosed by preferred embodiments and optional features, modificationand variations thereof may be resorted to by those skilled in the art,and that such modifications and variations are considered to be withinthe scope of this invention. All patents, patent applications,textbooks, and peer-reviewed publications described herein are herebyincorporated by reference in their entirety. Furthermore, where adefinition or use of a term in a reference, which is incorporated byreference herein is inconsistent or contrary to the definition of thatterm provided herein, the definition of that term provided hereinapplies and the definition of that term in the reference does not apply.Each of the narrower species and subgeneric groupings falling within thegeneric disclosure also forms part of the invention. This includes thegeneric description of the invention with a proviso or negativelimitation removing any subject matter from the genus, regardless ofwhether or not the excised material is specifically recited herein. Inaddition, where features are described in terms of Markush groups, thoseskilled in the art will recognize that the disclosure is also therebydescribed in terms of any individual member or subgroup of members ofthe Markush group. Further embodiments will become apparent from thefollowing claims.

Equivalents: Those skilled in the art will recognize, or be able toascertain using no more than routine experimentation, many equivalentsto the specific embodiments of the invention described herein. Suchequivalents are intended to be encompassed by the following claims. Allpublications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference into thespecification to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated herein by reference.

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1. A lipocalin mutein that is capable of binding lymphocyte-activationprotein 3 (LAG-3), wherein the mutein has at least 85% sequence identityto an amino acid sequence selected from the group consisting of SEQ IDNOs: 20-28, wherein the amino acid sequence of the mutein comprises thefollowing amino acid mutations in comparison with the linear polypeptidesequence of mature human tear lipocalin (SEQ ID NO: 1): Ser 14→Pro; Asp25→Ser; Phe 28→Asp; Lys 52→Arg; Met 55→Val; Ser 58→Asp; Ala 66→Asn; Ala79→Glu; Ala 86→Asp; Cys 101→Phe; Leu 105→Gly; Lys 108→Thr; Lys 114→Ala;Lys 121→Thr, and wherein the amino acid sequence of the mutein furthercomprises at least one of the following mutated amino acid residues incomparison with the linear polypeptide sequence of mature human tearlipocalin (SEQ ID NO: 1): Arg 26→Ser, Asp, Glu, or Ala; Met 31→Leu; Asn32→Thr; His 84→Tyr or Leu; His 106→Glu, Lys, or Pro; Val 110→Asn; Gly112→Val or Leu; Val 113→Ala or Leu.
 2. The lipocalin mutein of claim 1,wherein (i) the mutein is capable of binding LAG-3 with an affinitymeasured by K_(d) of about 250 nM or lower, about 50 nM or lower, about3 nM or lower, about 0.1 nM or lower, or about 0.05 nM or lower; (ii)the mutein binds LAG-3 with an EC₅₀ value of about 320 nM or lower,about 10 nM or lower, or about 0.2 nM or lower; (iii) the mutein iscross-reactive with both human LAG-3 and cynomolgus LAG-3; or (iv) themutein is capable of interfering with the binding of human LAG-3 tomajor histocompatibility complex (MHC) class II.
 3. The lipocalin muteinof claim 1, wherein the amino acid sequence of the mutein comprises atleast 20 of the following mutated amino acid residues in comparison withthe linear polypeptide sequence of mature human tear lipocalin (SEQ IDNO: 1): Ser 14→Pro; Asp 25→Ser; Arg 26→Ser, Asp, Glu, Ala, or Gly; Phe28→Asp; Met 31→Leu; Asn 32→Met or Thr; Lys 52→Arg; Met 55→Val; Ser58→Asp; Ala 66→Asn; Ala 79→Glu; His 84→Tyr or Leu; Ala 86→Asp; Cys101→Phe; Leu 105→Gly; His 106→Gln, Glu, Lys, or Pro; Lys 108→Thr; Val110→Gly or Asn; Gly 112→Met, Val or Leu; Val 113→Ala or Leu; Lys114→Ala; Lys 121→Thr.
 4. The lipocalin mutein of claim 1, wherein theamino acid sequence of the mutein comprises one of the following sets ofamino acid mutations in comparison with the linear polypeptide sequenceof mature human tear lipocalin (SEQ ID NO: 1): (a) Ser 14→Pro; Asp25→Ser; Arg 26→Asp; Phe 28→Asp; Asn 32→Thr; Lys 52→Arg; Met 55→Val; Ser58→Asp; Ala 66→Asn; Ala 79→Glu; His 84→Tyr; Ala 86→Asp; Cys 101→Phe; Leu105→Gly; Lys 108→Thr; Val 110→Gly; Gly 112→Met; Lys 114→Ala; and Lys121→Thr; (b) Ser 14→Pro; Asp 25→Ser; Arg 26→Glu; Phe 28→Asp; Met 31→Leu;Asn 32→Thr; Lys 52→Arg; Met 55→Val; Ser 58→Asp; Ala 66→Asn; Ala 79→Glu;His 84→Tyr; Ala 86→Asp; Cys 101→Phe; Leu 105→Gly; His 106→Gln; Lys108→Thr; Val 110→Gly; Gly 112→Met; Lys 114→Ala; and Lys 121→Thr; (c) Ser14→Pro; Asp 25→Ser; Arg 26→Glu; Phe 28→Asp; Asn 32→Thr; Lys 52→Arg; Met55→Val; Ser 58→Asp; Ala 66→Asn; Ala 79→Glu; His 84→Tyr; Ala 86→Asp; Cys101→Phe; Leu 105→Gly; His 106→Glu; Lys 108→Thr; Val 110→Gly; Gly112→Val; Lys 114→Ala; and Lys 121→Thr; (d) Ser 14→Pro; Asp 25→Ser; Arg26→Asp; Phe 28→Asp; Asn 32→Thr; Lys 52→Arg; Met 55→Val; Ser 58→Asp; Ala66→Asn; Ala 79→Glu; His 84→Tyr; Ala 86→Asp; Cys 101→Phe; Leu 105→Gly;His 106→Gln; Lys 108→Thr; Val 110→Gly; Gly 112→Leu; Lys 114→Ala; and Lys121→Thr; (e) Ser 14→Pro; Asp 25→Ser; Arg 26→Ser; Phe 28→Asp; Asn 32→Thr;Lys 52→Arg; Met 55→Val; Ser 58→Asp; Ala 66→Asn; Ala 79→Glu; His 84→Tyr;Ala 86→Asp; Cys 101→Phe; Leu 105→Gly; His 106→Gln; Lys 108→Thr; Val110→Gly; Gly 112→Met; Lys 114→Ala; and Lys 121→Thr; (f) Ser 14→Pro; Asp25→Ser; Arg 26→Ala; Phe 28→Asp; Asn 32→Thr; Lys 52→Arg; Met 55→Val; Ser58→Asp; Ala 66→Asn; Ala 79→Glu; His 84→Tyr; Ala 86→Asp; Cys 101→Phe; Leu105→Gly; His 106→Lys; Lys 108→Thr; Val 110→Gly; Gly 112→Met; Lys114→Ala; and Lys 121→Thr; (g) Ser 14→Pro; Asp 25→Ser; Phe 28→Asp; Asn32→Thr; Lys 52→Arg; Met 55→Val; Ser 58→Asp; Ala 66→Asn; Ala 79→Glu; Ala86→Asp; Cys 101→Phe; Leu 105→Gly; His 106→Gln; Lys 108→Thr; Val 110→Asn;Gly 112→Met; Val 113→Ala; Lys 114→Ala; and Lys 121→Thr; (h) Ser 14→Pro;Asp 25→Ser; Arg 26→Gly; Phe 28→Asp; Met 31→Leu; Asn 32→Thr; Lys 52→Arg;Met 55→Val; Ser 58→Asp; Ala 66→Asn; Ala 79→Glu; His 84→Tyr; Ala 86→Asp;Cys 101→Phe; Leu 105→Gly; His 106→Pro; Lys 108→Thr; Val 110→Gly; Gly112→Met; Lys 114→Ala; and Lys 121→Thr; or (i) Ser 14→Pro; Asp 25→Ser;Arg 26→Asp; Phe 28→Asp; Asn 32→Thr; Lys 52→Arg; Met 55→Val; Ser 58→Asp;Ala 66→Asn; Ala 79→Glu; His 84→Leu; Ala 86→Asp; Cys 101→Phe; Leu105→Gly; His 106→Gln; Lys 108→Thr; Val 110→Gly; Gly 112→Met; Val113→Leu; Lys 114→Ala; and Lys 121→Thr.
 5. The lipocalin mutein of claim1, wherein the amino acid sequence of the mutein comprises cysteineresidues at the sequence positions 61 and 153 of the linear polypeptidesequence of mature human tear lipocalin (SEQ ID NO: 1).
 6. The lipocalinmutein of claim 1, wherein the mutein comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 20-28 or a functionalfragment thereof.
 7. The lipocalin mutein claim 1, wherein the muteinhas at least 90%, at least 95%, at least 97.5% or at least 99% sequenceidentity to an amino acid sequence selected from the group consisting ofSEQ ID NOs: 20-28.
 8. The lipocalin mutein of claim 1, wherein themutein is conjugated to a compound selected from the group consisting ofan organic molecule, an enzyme label, a radioactive label, a coloredlabel, a fluorescent label, a chromogenic label, a luminescent label, ahapten, digoxigenin, biotin, a cytostatic agent, a toxin, a metalcomplex, a metal, colloidal gold, and a compound that extends the serumhalf-life of the mutein.
 9. The lipocalin mutein of claim 8, wherein thecompound that extends the serum half-life is selected from the groupconsisting of a polyalkylene glycol molecule, a polyethylene (PEG)glycol molecule, hydroxyethyl starch, an Fc part of an immunoglobulin, aC_(H)3 domain of an immunoglobulin, a C_(H)4 domain of animmunoglobulin, an albumin binding peptide, and an albumin bindingprotein.
 10. The lipocalin mutein of claim 1, wherein the mutein isfused at its N-terminus and/or its C-terminus to a fusion partner thatis an antibody, an antibody fragment, a protein, a protein domain, or apeptide.
 11. A nucleic acid molecule comprising a nucleotide sequenceencoding the lipocalin mutein of claim
 1. 12. A host cell containing anucleic acid molecule of claim
 11. 13. A method of producing a lipocalinmutein of claim 1, wherein the mutein is produced starting from thenucleic acid coding for the mutein or fragment thereof.
 14. A method ofbinding or detecting LAG-3 in a subject, comprising applying one or morelipocalin muteins of claim 1 or one or more compositions comprising suchmuteins.
 15. A method of stimulating an immune response in a subject,comprising applying one or more lipocalin muteins of claim 1 or one ormore compositions comprising such muteins.
 16. A method of inducing Tlymphocyte proliferation in a subject, comprising applying one or morelipocalin muteins of claim 1 or one or more compositions comprising suchmuteins.
 17. A method of interfering with the binding of human LAG-3 toMHC class II in a subject, comprising applying one or more lipocalinmuteins of claim 1 or one or more compositions comprising such muteins.18. A pharmaceutical composition comprising the lipocalin mutein ofclaim 1 and a pharmaceutically acceptable excipient.
 19. Animmunoconjugate or fusion protein comprising the lipocalin mutein, orfragment thereof, of claim 1 linked to a therapeutic agent.
 20. Adiagnostic or analytical kit comprising the lipocalin mutein of claim 1.